Streptococcus vaccine compositions and methods of using the same

ABSTRACT

The present invention provides methods and compositions for the stimulation of immune responses. In particular, the present invention provides immunogenic nanoemulsion compositions and methods of using the same for the induction of immune responses (e.g., innate and/or adaptive immune responses (e.g., for generation of host immunity against a bacterial species of the genus  Streptococcus  (e.g.,  Streptococcus pneumoniae ))). Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic and preventative medicine (e.g., vaccination)) and research applications.

This Application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/178,344 filed 14 May 2009, hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention provides methods and compositions for thestimulation of immune responses. In particular, the present inventionprovides immunogenic nanoemulsion compositions and methods of using thesame for the induction of immune responses (e.g., innate and/or adaptiveimmune responses (e.g., for generation of host immunity against abacterial species of the genus Streptococcus (e.g., Streptococcuspneumoniae))). Compositions and methods of the present invention finduse in, among other things, clinical (e.g. therapeutic and preventativemedicine (e.g., vaccination)) and research applications.

BACKGROUND

Immunization is a principal feature for improving the health of people.Despite the availability of a variety of successful vaccines againstmany common illnesses, infectious diseases remain a leading cause ofhealth problems and death. Significant problems inherent in existingvaccines include the need for repeated immunizations, and theineffectiveness of the current vaccine delivery systems for a broadspectrum of diseases.

In order to develop vaccines against pathogens that have beenrecalcitrant to vaccine development, and/or to overcome the failings ofcommercially available vaccines due to expense, complexity, andunderutilization, new methods of antigen presentation must be developedwhich will allow for fewer immunizations, more efficient usage, and/orfewer side effects to the vaccine.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for thestimulation of immune responses. In particular, the present inventionprovides immunogenic nanoemulsion compositions and methods of using thesame for the induction of immune responses (e.g., innate and/or adaptiveimmune responses (e.g., for generation of host immunity against abacterial species of the genus Streptococcus (e.g., Streptococcuspneumoniae))). Compositions and methods of the present invention finduse in, among other things, clinical (e.g. therapeutic and preventativemedicine (e.g., vaccination)) and research applications.

In some embodiments, the present invention provides an immunogeniccomposition comprising a nanoemulsion and one or more Streptococcusantigens (e.g., Streptococcus pneumoniae antigens). In some embodiments,the nanoemulsion comprises an aqueous phase, an oil phase, and asolvent. The present invention is not limited by the type ofnanoemulsion composition. Indeed, a variety of nanoemulsion compositionsfind use in the present invention including, but not limited to, thosedescribed herein. The present invention is not limited by the one ormore Streptococcus antigens utilized in the immunogenic compositions andmethods of the invention. Indeed, a variety of Streptococcus antigensmay be utilized including, but not limited to, Streptococcal bacteriainactivated and/or killed by nanoemulsion (NE), killed and/orinactivated Streptococcus bacteria (e.g., via mixing with alcohol (e.g.,ethanol)), whole cell lysates of a Streptococcus bacteria, one or moreisolated, purified and/or recombinant Streptococcus proteins and/orprotein fragments, or other type of Streptococcus antigen describedherein. In a preferred embodiment, the Streptococcus antigen is killedStreptococcus pneumoniae (e.g., (See, e.g., Malley et al., (2001)Infect. Immun. 69, 4870-4873; Malley et al., (2004) Infect. Immun. 72,4290-4292)). In some embodiments, a Streptococcus antigen compositioncomprises one or more adjuvants (e.g., cholera toxin (CT)). The presentinvention is not limited by the strain and/or serotype of Streptococcuspneumoniae utilized. A number of strains and/or serotypes ofStreptococcus pneumoniae are described herein, each of which finds usein an immunogenic composition comprising a nanoemulsion and one or moreStreptococcus antigens. In some embodiments, the immunogenic compositioncomprises nanoemulsion inactivated bacteria of the genus Streptococcus(e.g., Streptococcus pneumoniae). In some embodiments, the nanoemulsionis W₈₀5EC, although the present invention is not so limited. Forexample, in some embodiments, the nanoemulsion is selected from one ofthe nanoemulsion formulations described herein. In some embodiments, thecomposition comprises between 1-50% nanoemulsion solution, althoughgreater and lesser amounts also find use in the invention. For example,in some embodiments, the immunogenic composition comprises about1.0%-10%, about 10%-20%, about 20%-30%, about 30%-40%, about 40%-50%,about 50%-60% or more nanoemulsion solution. In some embodiments, theimmunogenic composition comprises about 10% nanoemulsion solution. Insome embodiments, the immunogenic composition comprises about 15%nanoemulsion solution. In some embodiments, the immunogenic compositioncomprises about 20% nanoemulsion solution. In some embodiments, theimmunogenic composition comprises about 12% nanoemulsion solution. Insome embodiments, the immunogenic composition comprises about 8%nanoemulsion solution. In some embodiments, the immunogenic compositioncomprises about 5% nanoemulsion solution. In some embodiments, theimmunogenic composition comprises about 2% nanoemulsion solution. Insome embodiments, the immunogenic composition comprises about 1%nanoemulsion solution. In some embodiments, an immunogenic composition(e.g., that is administered to a subject in order to generate aStreptococcus specific immune response in the subject) comprises 10⁶colony forming units (CFU) of killed Streptococcus (e.g., 10⁶ CFU ofStreptococcus pneumoniae bacteria prior to killing/inactivation of thebacteria), although greater (e.g., about 4×10⁶ CFU, 8×10⁶ CFU, 1×10⁷CFU, 2×10⁷ CFU, 4×10⁷ CFU, 8×10⁷ CFU, 1×10⁸ CFU, 1×10⁹ CFU, or more CFUof killed Streptococcus) and lesser (e.g., about 1×10⁶ CFU, 5×10⁵ CFU,1×10⁵ CFU, 5×10⁴ CFU, 1×10⁴ CFU, 5×10³ CFU, 1×10³ CFU or fewer CFU ofkilled Streptococcus) amounts may also be utilized. In some embodiments,the composition is stable (e.g., at room temperature (e.g., for 12hours, one day, two days, three days, four days, a week, two weeks,three weeks, a month, two months, three months, four months, fivemonths, six months, 9 months, a year or more). In some embodiments, theimmunogenic composition comprises a pharmaceutically acceptable carrier.The present invention is not limited to any particular pharmaceuticallyacceptable carrier. Indeed, any suitable carrier may be utilizedincluding but not limited to those described herein. In someembodiments, the immunogenic composition further comprises an adjuvant.The present invention is not limited to any particular adjuvant and anyone or more adjuvants described herein find use in a composition of theinvention including but not limited to adjuvants that skew toward a Th1and/or Th2 type immune responses described herein. In some embodiments,the immunogen comprises a Streptococcus product (e.g., including, butnot limited to, a protein, peptide, polypeptide, nucleic acid,polysaccharide, or a membrane component derived from the Streptococcus).In some embodiments, the immunogen and the nanoemulsion are combined ina single vessel.

In some embodiments, the present invention provides a method of inducingan immune response to Streptococcus (e.g., Streptococcus pneumoniae) ina subject comprising: providing a subject and an immunogenic compositioncomprising a nanoemulsion and an immunogen, wherein the immunogencomprises a Streptococcus (e.g., Streptococcus pneumoniae) antigen andadministering the composition to the subject under conditions such thatthe subject generates a Streptococcus (e.g., Streptococcus pneumoniae)specific immune response. The present invention is not limited by theroute chosen for administration of a composition of the presentinvention. In some preferred embodiments, administering the immunogeniccomposition comprises contacting a mucosal surface of the subject withthe composition. In some embodiments, the mucosal surface comprisesnasal mucosa. In some embodiments, inducing an immune response inducesimmunity to Streptococcus (e.g., Streptococcus pneumoniae) in thesubject. In some embodiments, the immunity comprises systemic immunity.In some embodiments, the immunity comprises mucosal immunity. In someembodiments, the immune response comprises altered (e.g., enhanced)cytokine expression in the subject. In some embodiments, the immuneresponse comprises an IgG response (e.g., a systemic IgG response) tothe Streptococcus (e.g., Streptococcus pneumoniae) in the subject. Insome embodiments, the Streptococcus (e.g., Streptococcus pneumoniae)antigenic composition is administered to the subject under conditionssuch that between about 10⁵ and 10⁸ colony forming units (CFU) ofStreptococcus (e.g., Streptococcus pneumoniae) is present in a doseadministered to the subject, although greater (e.g., about 10⁹,10¹⁰,10¹¹, 10¹², or more) and lesser (e.g., about 10⁴, 10³, 10² orfewer) CFU of Streptococcus (e.g., Streptococcus pneumoniae) (e.g.,killed whole Streptococcus pneumoniae) may also be utilized. In someembodiments, a nanoemulsion solution is utilized to inactivate theStreptococcus (e.g., Streptococcus pneumoniae). In some embodiments, thenanoemulsion comprises W₈₀5EC. In some embodiments, the immunityprotects the subject from displaying signs or symptoms of disease causedby Streptococcus (e.g., Streptococcus pneumoniae). In some embodiments,the immunity protects the subject from challenge with a subsequentexposure to live Streptococcus (e.g., Streptococcus pneumoniae). In someembodiments, the composition further comprises an adjuvant. In someembodiments, the subject is a human. In some embodiments, inducing animmune response induces immunity to the Streptococcus (e.g.,Streptococcus pneumoniae) in the subject. In some embodiments, inducingimmunity to Streptococcus (e.g., Streptococcus pneumoniae) comprisessystemic immunity. In some embodiments, immunity comprises mucosalimmunity. In some embodiments, the immune response comprises altered(e.g., increased) cytokine expression in the subject. In someembodiments, the immune response comprises a systemic IgG response tothe immunogen. In some embodiments, the immune response comprises amucosal IgA response to the immunogen. In some embodiments, each dosecomprises an amount of Streptococcus (e.g., Streptococcus pneumoniae)antigen sufficient to generate an immune response to the Streptococcus(e.g., Streptococcus pneumoniae). An effective amount of theStreptococcus (e.g., Streptococcus pneumoniae) antigen is a dose thatneed not be quantified, as long as the amount of Streptococcus (e.g.,Streptococcus pneumoniae) antigen generates an immune response in asubject when administered to the subject. In some embodiments, when ananoemulsion of the present invention is utilized to inactivateStreptococcus (e.g., Streptococcus pneumoniae), it is expected that eachdose (e.g., administered to a subject to induce and immune response))comprises between 10 and 10¹⁰ CFU of Streptococcus (e.g., Streptococcuspneumoniae) per dose; in some embodiments, each dose comprises between10⁵ and 10⁸ CFU of Streptococcus (e.g., Streptococcus pneumoniae) perdose; in some embodiments, each dose comprises between 10³ and 10⁵ CFUof Streptococcus (e.g., Streptococcus pneumoniae) per dose; in someembodiments, each dose comprises between 10⁵ and 10⁸ CFU ofStreptococcus (e.g., Streptococcus pneumoniae) per dose; in someembodiments, each dose comprises 10⁵ CFU of Streptococcus (e.g.,Streptococcus pneumoniae) per dose; in some embodiments, each dosecomprises 10⁶ CFU of Streptococcus (e.g., Streptococcus pneumoniae) perdose; and in some embodiments, each dose comprises 10⁷ CFU ofStreptococcus (e.g., Streptococcus pneumoniae) per dose. In someembodiments, each dose comprises more than 10⁸ CFU of Streptococcus(e.g., Streptococcus pneumoniae) per dose. In some preferredembodiments, each dose comprises 10⁸ CFU of Streptococcus (e.g.,Streptococcus pneumoniae) per dose.

The present invention is not limited to any specific nanoemulsioncomposition. Indeed, a variety of nanoemulsion compositions aredescribed herein that find use in the present invention. Similarly, thepresent invention is not limited to a particular oil present in thenanoemulsion. A variety of oils are contemplated, including, but notlimited to, soybean, avocado, squalene, olive, canola, corn, rapeseed,safflower, sunflower, fish, flavor, and water insoluble vitamins. Thepresent invention is also not limited to a particular solvent. A varietyof solvents are contemplated including, but not limited to, an alcohol(e.g., including, but not limited to, methanol, ethanol, propanol, andoctanol), glycerol, polyethylene glycol, and an organic phosphate basedsolvent. Nanoemulsion components including oils, solvents and others aredescribed in further detail below.

In some embodiments, the emulsion further comprises a surfactant. Thepresent invention is not limited to a particular surfactant. A varietyof surfactants are contemplated including, but not limited to, nonionicand ionic surfactants (e.g., TRITON X-100; TWEEN 20; and TYLOXAPOL).

In certain embodiments, the emulsion further comprises a cationichalogen containing compound. The present invention is not limited to aparticular cationic halogen containing compound. A variety of cationichalogen containing compounds are contemplated including, but not limitedto, cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides, andtetradecyltrimethylammonium halides. The present invention is also notlimited to a particular halide. A variety of halides are contemplatedincluding, but not limited to, halide selected from the group consistingof chloride, fluoride, bromide, and iodide.

In still further embodiments, the emulsion further comprises aquaternary ammonium containing compound. The present invention is notlimited to a particular quaternary ammonium containing compound. Avariety of quaternary ammonium containing compounds are contemplatedincluding, but not limited to, Alkyl dimethyl benzyl ammonium chloride,

dialkyl dimethyl ammonium chloride, n-Alkyl dimethyl benzyl ammoniumchloride, n-Alkyl dimethyl ethylbenzyl ammonium chloride, Dialkyldimethyl ammonium chloride, and n-Alkyl dimethyl benzyl ammoniumchloride.

In some embodiments, the present invention provides a vaccine comprisingan immunogenic composition comprising Streptococcus (e.g., Streptococcuspneumoniae) antigen (e.g., killed and/or inactivated whole cellStreptococcus (e.g., Streptococcus pneumoniae)). In some embodiments,the invention provides a kit comprising a vaccine, the vaccinecomprising a nanoemulsion and immunogenic composition comprisingStreptococcus (e.g., Streptococcus pneumoniae) antigen, the emulsioncomprising an aqueous phase, an oil phase, and a solvent. In someembodiments, the kit further comprises instructions for using the kitfor vaccinating a subject against the Streptococcus (e.g., Streptococcuspneumoniae).

In still further embodiments, the present invention provides a method ofinducing immunity to one or more bacteria of the genus Streptococcus(e.g., Streptococcus pneumoniae) comprising providing an emulsioncomprising an aqueous phase, an oil phase, and a solvent; and one ormore Streptococcus (e.g., Streptococcus pneumoniae) antigens; combiningthe emulsion with the one or more Streptococcus (e.g., Streptococcuspneumoniae) antigens to generate a vaccine composition; andadministering the vaccine composition to a subject. In some embodiments,administering comprises contacting the vaccine composition with amucosal surface of the subject. For example, in some preferredembodiments, administering comprises intranasal administration. In somepreferred embodiments, the administering occurs under conditions suchthat the subject generates immunity to the one or more bacteria of thegenus Streptococcus (e.g., Streptococcus pneumoniae) (e.g., viagenerating humoral immune responses to the one or more antigens).

The present invention is not limited by the nature of the immuneresponse generated (e.g., post administration of an immunogeniccomposition. Indeed, a variety of immune responses may be generated andmeasured in a subject administered a composition of the presentinvention including, but not limited to, activation, proliferation ordifferentiation of cells of the immune system (e.g., B cells, T cells,dendritic cells, antigen presenting cells (APCs), macrophages, naturalkiller (NK) cells, etc.); up-regulated or down-regulated expression ofmarkers and cytokines; stimulation of IgA, IgM, and/or IgG titers;splenomegaly (e.g., increased spleen cellularity); hyperplasia, mixedcellular infiltrates in various organs, and/or other responses (e.g., ofcells) of the immune system that can be assessed with respect to immunestimulation known in the art. In some embodiments, administeringcomprises contacting a mucosal surface of the subject with thecomposition. The present invention is not limited by the mucosal surfacecontacted. In some preferred embodiments, the mucosal surface comprisesnasal mucosa. In some embodiments, administering comprises parenteraladministration. The present invention is not limited by the route chosenfor administration of a composition of the present invention. In someembodiments, inducing an immune response induces immunity to the one ormore bacteria of the genus Streptococcus (e.g., Streptococcuspneumoniae) in the subject. In some embodiments, the immunity comprisessystemic immunity. In some embodiments, the immunity comprises mucosalimmunity. In some embodiments, the immune response comprises altered(e.g., increased) cytokine expression in the subject. In someembodiments, the immune response comprises a systemic IgG response. Insome embodiments, the immune response comprises a mucosal IgA response.In some embodiments, the composition comprises a 15% nanoemulsionsolution. However, the present invention is not limited to this amount(e.g., percentage) of nanoemusion. For example, in some embodiments, acomposition comprises less than 10% nanoemulsion (e.g., 9%, 8%, 7%, 6%,5%, 4%, 3%, 2% or 1%). In some embodiments, a composition comprises morethan 10% nanoemulsion (e.g., 15%, 20%, 25%, 30%, 35%, 40%. 45%, 50%, 60%or more). In some embodiments, a composition of the present inventioncomprises any of the nanoemulsions described herein. In someembodiments, the nanoemulsion comprises W₂₀5EC. In some preferredembodiments, the nanoemulsion comprises W₈₀5EC. In some embodiments, thenanoemulsion is X8P. In some embodiments, immunity protects the subjectfrom displaying signs or symptoms of disease caused by a bacteria of thegenus Streptococcus (e.g., Streptococcus pneumoniae). In someembodiments, immunity protects the subject from challenge with asubsequent exposure to a live bacterium of the genus Streptococcus(e.g., Streptococcus pneumoniae). In some embodiments, the compositionfurther comprises an adjuvant. The present invention is not limited bythe type of adjuvant utilized. In some embodiments, the adjuvant ischolera toxin (CT). In some embodiments, the adjuvant is monophosphoryllipid A and/or a CpG oligonucleotide. A number of other adjuvants thatfind use in the present invention are described herein. In someembodiments, the subject is a human. In some embodiments, the immunityprotects the subject from displaying signs or symptoms of a infectionwith a bacteria of the genus Streptococcus (e.g., Streptococcuspneumoniae). In some embodiments, immunity reduces the risk ofinfection, disease, and/or sickness upon one or more exposures to abacteria of the genus Streptococcus (e.g., Streptococcus pneumoniae).

DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects and embodiments of thepresent invention. The invention may be better understood by referenceto one or more of these figures in combination with the description ofspecific embodiments presented herein.

FIG. 1 shows the preparation of various single dose formulations ofvaccines using stock WCPAg.

FIG. 2 shows the distribution of anti-S. pneumoniae antibody titers at 8weeks after two vaccinations.

FIG. 3 shows the distribution of anti-S. pneumoniae antibody titers at10 weeks after three vaccinations of A) 7.5 μl or B) 0.1 μl of S.pneumoniae antigen.

FIG. 4 shows serum anti-S. pneumoniae IgG antibodies in CD-1 micemeasured at 11 weeks after primary immunization presented as endpointtitters (+/−sd).

FIG. 5 shows results of a post-mortem nasalpharyngeal lavage that wasperformed at 12 weeks (1 week following challenge). The lavagent wasplated on selective media and wildtype 6B S. pneumoniae were enumerated.WCAg=whole cell antigen. Data is presented as endpoint titters (+/−sd).

FIG. 6 shows the S. pneumoniae colony count using plate culture afterincubation with W₈₀5EC.

FIG. 7 identifies subjects and the various compositions administered tothe subjects for experiments conducted during the development ofembodiments of the invention.

FIG. 8 shows serum anti-S. pneumoniae IgG antibodies in CD-1 micemeasured at 8 weeks after primary immunization. Data presented asendpoint titters (+/−sd).

FIG. 9 shows results of a post-mortem nasalpharyngeal lavage performedat 9 weeks (1 week following challenge. The lavagent was plated onselective media and wildtype 6B S. pneumoniae were enumerated. Datapresented as endpoint titters (+/−sd).

FIG. 10 shows western blots of pneumococcal antigens probed with eitherserum from mice vaccinated with NE-S. pneumo (left) or Alum-S. pneumo(right). Lane 1 and 5 represent the molecular weight ladder. Lane 2represents S. pneumoniae inactivated with NE. Lane 3 represents ethanolinactivated S. pneumoniae. Lane 4 represents wildtype 6B antigen.

GENERAL DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for thestimulation of immune responses. In particular, the present inventionprovides immunogenic nanoemulsion compositions and methods of using thesame for the induction of immune responses (e.g., innate and/or adaptiveimmune responses (e.g., for generation of host immunity against abacterial species of the genus Streptococcus (e.g., Streptococcuspneumoniae))). Compositions and methods of the present invention finduse in, among other things, clinical (e.g. therapeutic and preventativemedicine (e.g., vaccination)) and research applications.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, nanoemulsion (NE)compositions stabilize and/or preserve (e.g., for antigen presentation)important antigenic epitopes (e.g., recognizable by a subject's immunesystem) of a bacteria of the genus Streptococcus (e.g., Streptococcuspneumoniae)) (e.g., stabilize and/or preserve hydrophobicand/hydrophilic components in the oil and water interface of theemulsion (e.g., thereby providing one or more immunogens (e.g.,stabilized antigens) against which a subject can mount an immuneresponse). In other embodiments, because NE formulations penetrate themucosa through pores, they may carry antigens/immunogens to thesubmucosal location of dendritic cells (e.g., thereby initiating and/orstimulating an immune response). Although an understanding of themechanism is not necessary to practice the present invention and thepresent invention is not limited to any particular mechanism of action,in some embodiments, combining a NE and Streptococcus (e.g.,Streptococcus pneumoniae)) antigen stabilizes and/or preservesStreptococcus (e.g., Streptococcus pneumoniae)) immunogens and providesa proper immunogenic material for generation of an immune response.Dendritic cells avidly phagocytose nanoemulsion (NE) oil droplets andthis provides one mechanism to internalize immunogens (e.g., antigenicproteins or peptide fragments thereof of Streptococcus (e.g.,Streptococcus pneumoniae) for antigen presentation. While other vaccinesrely on inflammatory toxins or other immune stimuli for adjuvantactivity (See, e.g., Holmgren and Czerkinsky, Nature Med. 2005, 11;45-53), NEs have not been shown to be inflammatory when placed on theskin or mucous membranes in studies on animals and in humans. Thus,although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, a compositioncomprising a NE of the present invention (e.g., a composition comprisingNE and one or more Streptococcus (e.g., Streptococcus pneumoniae))antigens may act as a “physical” adjuvant (e.g., that transports and/orpresents immunogenic compositions (e.g., peptides and/or antigens ofStreptococcus (e.g., Streptococcus pneumoniae)) to the immune system. Insome embodiments, mucosal administration of a composition of the presentinvention generates mucosal (e.g., signs of mucosal immunity (e.g.,generation of IgA antibody titers)) as well as systemic immunity.

Both cellular and humoral immunity play a role in protection againstmultiple pathogens and both can be induced with the NE formulations ofthe present invention. In some embodiments, administration (e.g.,mucosal administration) of a composition of the present invention to asubject results in the induction of both humoral (e.g., development ofspecific antibodies) and cellular (e.g., cytotoxic T lymphocyte) immuneresponses (e.g., against Streptococcus (e.g., Streptococcuspneumoniae)). In some embodiments, a composition of the presentinvention (e.g., immunogenic composition comprising NE and Streptococcus(e.g., Streptococcus pneumoniae)) antigen is used as a vaccine (e.g., anRSV vaccine).

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “microorganism” refers to any species or typeof microorganism, including but not limited to, bacteria, viruses,archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms.The term microorganism encompasses both those organisms that are in andof themselves pathogenic to another organism (e.g., animals, includinghumans, and plants) and those organisms that produce agents that arepathogenic to another organism, while the organism itself is notdirectly pathogenic or infective to the other organism.

As used herein the term “pathogen,” and grammatical equivalents, refersto an organism (e.g., biological agent), including microorganisms, thatcauses a disease state (e.g., infection, pathologic condition, disease,etc.) in another organism (e.g., animals and plants) by directlyinfecting the other organism, or by producing agents that causes diseasein another organism (e.g., bacteria that produce pathogenic toxins andthe like). “Pathogens” include, but are not limited to, viruses,bacteria, archaea, fungi, protozoans, mycoplasma, prions, and parasiticorganisms.

The terms “bacteria” and “bacterium” refer to all prokaryotic organisms,including those within all of the phyla in the Kingdom Procaryotae. Itis intended that the term encompass all microorganisms considered to bebacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, andRickettsia. All forms of bacteria are included within this definitionincluding cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.

As used herein, the term Streptococcus refers to a genus of sphericalGram positive bacteria belonging to the phylum Firmicutes and the lacticacid bacteria group. In general, Streptococci are oxidase-negative andcatalase-negative, and many are facultative anaerobes. In general,individual species of Streptococcus are classified based on theirhemolytic properties. Alpha hemolysis is caused by a reduction of ironin hemoglobin, giving it a greenish color on blood agar. Beta-onlyhemolysis is complete rupture of red blood cells, giving distinct, wide,clear areas around bacterial colonies on blood agar. Other streptococciare labeled as gamma hemolytic.

As used herein, the term “fungi” is used in reference to eukaryoticorganisms such as molds and yeasts, including dimorphic fungi.

As used herein the terms “disease” and “pathologic condition” are usedinterchangeably, unless indicated otherwise herein, to describe adeviation from the condition regarded as normal or average for membersof a species or group (e.g., humans), and which is detrimental to anaffected individual under conditions that are not inimical to themajority of individuals of that species or group. Such a deviation canmanifest as a state, signs, and/or symptoms (e.g., diarrhea, nausea,fever, pain, blisters, boils, rash, immune suppression, inflammation,etc.) that are associated with any impairment of the normal state of asubject or of any of its organs or tissues that interrupts or modifiesthe performance of normal functions. A disease or pathological conditionmay be caused by or result from contact with a microorganism (e.g., apathogen or other infective agent (e.g., a virus or bacteria)), may beresponsive to environmental factors (e.g., malnutrition, industrialhazards, and/or climate), may be responsive to an inherent defect of theorganism (e.g., genetic anomalies) or to combinations of these and otherfactors.

The terms “host” or “subject,” as used herein, refer to an individual tobe treated by (e.g., administered) the compositions and methods of thepresent invention. Subjects include, but are not limited to, mammals(e.g., murines, simians, equines, bovines, porcines, canines, felines,and the like), and most preferably includes humans. In the context ofthe invention, the term “subject” generally refers to an individual whowill be administered or who has been administered one or morecompositions of the present invention (e.g., a composition for inducingan immune response).

As used herein, the terms “inactivating,” “inactivation” and grammaticalequivalents, when used in reference to a microorganism (e.g., a pathogen(e.g., a bacterium or a virus)), refer to the killing, elimination,neutralization and/or reducing of the capacity of the microorganism(e.g., a pathogen (e.g., a bacterium or a virus)) to infect and/or causea pathological response and/or disease in a host. For example, in someembodiments, the present invention provides a composition comprisingnanoemulsion (NE)-inactivated Staphylococci. Accordingly, as referred toherein, compositions comprising “NE-inactivated Staphylococci,”“NE-killed Staphylococci,” NE-neutralized Staphylococci” or grammaticalequivalents refer to compositions that, when administered to a subject,are characterized by the absence of, or significantly reduced presenceof, Staphylococci replication (e.g., over a period of time (e.g., over aperiod of days, weeks, months, or longer)) within the host.

As used herein, the term “fusigenic” is intended to refer to an emulsionthat is capable of fusing with the membrane of a microbial agent (e.g.,a bacterium or bacterial spore). Specific examples of fusigenicemulsions are described herein.

As used herein, the term “lysogenic” refers to an emulsion (e.g., ananoemulsion) that is capable of disrupting the membrane of a microbialagent (e.g., a virus (e.g., viral envelope) or a bacterium or bacterialspore). In preferred embodiments of the present invention, the presenceof a lysogenic and a fusigenic agent in the same composition produces anenhanced inactivating effect compared to either agent alone. Methods andcompositions (e.g., for inducing an immune response (e.g., used as avaccine) using this improved antimicrobial composition are described indetail herein.

The term “emulsion,” as used herein, includes classic oil-in-water orwater in oil dispersions or droplets, as well as other lipid structuresthat can form as a result of hydrophobic forces that drive apolarresidues (e.g., long hydrocarbon chains) away from water and drive polarhead groups toward water, when a water immiscible oily phase is mixedwith an aqueous phase. These other lipid structures include, but are notlimited to, unilamellar, paucilamellar, and multilamellar lipidvesicles, micelles, and lamellar phases. Similarly, the term“nanoemulsion,” as used herein, refers to oil-in-water dispersionscomprising small lipid structures. For example, in some embodiments, thenanoemulsions comprise an oil phase having droplets with a mean particlesize of approximately 0.1 to 5 microns (e.g., about 150, 200, 250, 300,350, 400, 450, 500 nm or larger in diameter), although smaller andlarger particle sizes are contemplated. The terms “emulsion” and“nanoemulsion” are often used herein, interchangeably, to refer to thenanoemulsions of the present invention.

As used herein, the terms “contact,” “contacted,” “expose,” and“exposed,” when used in reference to a nanoemulsion and a livemicroorganism, refer to bringing one or more nanoemulsions into contactwith a microorganism (e.g., a pathogen) such that the nanoemulsioninactivates the microorganism or pathogenic agent, if present. Thepresent invention is not limited by the amount or type of nanoemulsionused for microorganism inactivation. A variety of nanoemulsion that finduse in the present invention are described herein and elsewhere (e.g.,nanoemulsions described in U.S. Pat. Apps. 20020045667 and 20040043041,and U.S. Pat. Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, eachof which is incorporated herein by reference in its entirety for allpurposes). Ratios and amounts of nanoemulsion (e.g., sufficient forinactivating the microorganism (e.g., virus inactivation)) andmicroorganisms (e.g., sufficient to provide an antigenic composition(e.g., a composition capable of inducing an immune response)) arecontemplated in the present invention including, but not limited to,those described herein.

The term “surfactant” refers to any molecule having both a polar headgroup, which energetically prefers solvation by water, and a hydrophobictail that is not well solvated by water. The term “cationic surfactant”refers to a surfactant with a cationic head group. The term “anionicsurfactant” refers to a surfactant with an anionic head group.

The terms “Hydrophile-Lipophile Balance Index Number” and “HLB IndexNumber” refer to an index for correlating the chemical structure ofsurfactant molecules with their surface activity. The HLB Index Numbermay be calculated by a variety of empirical formulas as described, forexample, by Meyers, (See, e.g., Meyers, Surfactant Science andTechnology, VCH Publishers Inc., New York, pp. 231-245 (1992)),incorporated herein by reference. As used herein where appropriate, theHLB Index Number of a surfactant is the HLB Index Number assigned tothat surfactant in McCutcheon's Volume 1: Emulsifiers and DetergentsNorth American Edition, 1996 (incorporated herein by reference). The HLBIndex Number ranges from 0 to about 70 or more for commercialsurfactants. Hydrophilic surfactants with high solubility in water andsolubilizing properties are at the high end of the scale, whilesurfactants with low solubility in water that are good solubilizers ofwater in oils are at the low end of the scale.

As used herein the term “interaction enhancers” refers to compounds thatact to enhance the interaction of an emulsion with a microorganism(e.g., with a cell wall of a bacteria (e.g., a Gram negative bacteria)or with a viral envelope (e.g., Vaccinia virus envelope)). Contemplatedinteraction enhancers include, but are not limited to, chelating agents(e.g., ethylenediaminetetraacetic acid (EDTA),ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), and the like)and certain biological agents (e.g., bovine serum albumin (BSA) and thelike).

The terms “buffer” or “buffering agents” refer to materials, that whenadded to a solution, cause the solution to resist changes in pH.

The terms “reducing agent” and “electron donor” refer to a material thatdonates electrons to a second material to reduce the oxidation state ofone or more of the second material's atoms.

The term “monovalent salt” refers to any salt in which the metal (e.g.,Na, K, or Li) has a net 1+ charge in solution (i.e., one more protonthan electron).

The term “divalent salt” refers to any salt in which a metal (e.g., Mg,Ca, or Sr) has a net 2+ charge in solution.

The terms “chelator” or “chelating agent” refer to any materials havingmore than one atom with a lone pair of electrons that are available tobond to a metal ion.

The term “solution” refers to an aqueous or non-aqueous mixture.

As used herein, the term “a composition for inducing an immune response”refers to a composition that, once administered to a subject (e.g.,once, twice, three times or more (e.g., separated by weeks, months oryears)), stimulates, generates and/or elicits an immune response in thesubject (e.g., resulting in total or partial immunity to a microorganism(e.g., pathogen) capable of causing disease). In preferred embodimentsof the invention, the composition comprises a nanoemulsion and animmunogen. In further preferred embodiments, the composition comprisinga nanoemulsion and an immunogen comprises one or more other compounds oragents including, but not limited to, therapeutic agents,physiologically tolerable liquids, gels, carriers, diluents, adjuvants,excipients, salicylates, steroids, immunosuppressants, immunostimulants,antibodies, cytokines, antibiotics, binders, fillers, preservatives,stabilizing agents, emulsifiers, and/or buffers. An immune response maybe an innate (e.g., a non-specific) immune response or a learned (e.g.,acquired) immune response (e.g. that decreases the infectivity,morbidity, or onset of mortality in a subject (e.g., caused by exposureto a pathogenic microorganism) or that prevents infectivity, morbidity,or onset of mortality in a subject (e.g., caused by exposure to apathogenic microorganism)). Thus, in some preferred embodiments, acomposition comprising a nanoemulsion and an immunogen is administeredto a subject as a vaccine (e.g., to prevent or attenuate a disease(e.g., by providing to the subject total or partial immunity against thedisease or the total or partial attenuation (e.g., suppression) of asign, symptom or condition of the disease.

As used herein, the term “adjuvant” refers to any substance that canstimulate an immune response (e.g., a mucosal immune response). Someadjuvants can cause activation of a cell of the immune system (e.g., anadjuvant can cause an immune cell to produce and secrete a cytokine).Examples of adjuvants that can cause activation of a cell of the immunesystem include, but are not limited to, the nanoemulsion formulationsdescribed herein, saponins purified from the bark of the Q. saponariatree, such as QS21 (a glycolipid that elutes in the 21st peak with HPLCfractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.);poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); cholera toxin (CT), and Leishmaniaelongation factor (a purified Leishmania protein; Corixa Corporation,Seattle, Wash.). Traditional adjuvants are well known in the art andinclude, for example, aluminum phosphate or hydroxide salts (“alum”). Insome embodiments, compositions of the present invention (e.g.,comprising HIV or an immunogenic epitope thereof (e.g., gp120)) areadministered with one or more adjuvants (e.g., to skew the immuneresponse towards a Th1 and/or Th2 type response).

As used herein, the term “an amount effective to induce an immuneresponse” (e.g., of a composition for inducing an immune response),refers to the dosage level required (e.g., when administered to asubject) to stimulate, generate and/or elicit an immune response in thesubject. An effective amount can be administered in one or moreadministrations (e.g., via the same or different route), applications ordosages and is not intended to be limited to a particular formulation oradministration route.

As used herein, the term “under conditions such that said subjectgenerates an immune response” refers to any qualitative or quantitativeinduction, generation, and/or stimulation of an immune response (e.g.,innate or acquired).

A used herein, the term “immune response” refers to a response by theimmune system of a subject. For example, immune responses include, butare not limited to, a detectable alteration (e.g., increase) inToll-like receptor (TLR) activation, lymphokine (e.g., cytokine (e.g.,Th1 or Th2 type cytokines) or chemokine) expression and/or secretion,macrophage activation, dendritic cell activation, T cell activation(e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cellactivation (e.g., antibody generation and/or secretion). Additionalexamples of immune responses include binding of an immunogen (e.g.,antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducinga cytotoxic T lymphocyte (“CTL”) response, inducing a B cell response(e.g., antibody production), and/or T-helper lymphocyte response, and/ora delayed type hypersensitivity (DTH) response against the antigen fromwhich the immunogenic polypeptide is derived, expansion (e.g., growth ofa population of cells) of cells of the immune system (e.g., T cells, Bcells (e.g., of any stage of development (e.g., plasma cells), andincreased processing and presentation of antigen by antigen presentingcells. An immune response may be to immunogens that the subject's immunesystem recognizes as foreign (e.g., non-self antigens frommicroorganisms (e.g., pathogens), or self-antigens recognized asforeign). Thus, it is to be understood that, as used herein, “immuneresponse” refers to any type of immune response, including, but notlimited to, innate immune responses (e.g., activation of Toll receptorsignaling cascade) cell-mediated immune responses (e.g., responsesmediated by T cells (e.g., antigen-specific T cells) and non-specificcells of the immune system) and humoral immune responses (e.g.,responses mediated by B cells (e.g., via generation and secretion ofantibodies into the plasma, lymph, and/or tissue fluids). The term“immune response” is meant to encompass all aspects of the capability ofa subject's immune system to respond to antigens and/or immunogens(e.g., both the initial response to an immunogen (e.g., a pathogen) aswell as acquired (e.g., memory) responses that are a result of anadaptive immune response).

As used herein, the terms “toll receptors” and “TLRs” refer to a classof receptors (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR 11) that recognize special patterns of pathogens,termed pathogen-associated molecular patterns (See, e.g., Janeway andMedzhitov, (2002) Annu. Rev. Immunol. 20, 197-216). These receptors areexpressed in innate immune cells (e.g., neutrophils, monocytes,macrophages, dendritic cells) and in other types of cells such asendothelial cells. Their ligands include bacterial products such as LPS,peptidoglycans, lipopeptides, and CpG DNA. TLRs are receptors that bindto exogenous ligands and mediate innate immune responses leading to theelimination of invading microbes. The TLR-triggered signaling pathwayleads to activation of transcription factors including NFkB, which isimportant for the induced expression of proinflammatory cytokines andchemokines. TLRs also interact with each other. For example, TLR2 canform functional heterodimers with TLR1 or TLR6. The TLR2/1 dimer hasdifferent ligand binding profile than the TLR2/6 dimer (Ozinsky et al.,2000). In some embodiments, a nanoemulsion adjuvant activates cellsignaling through a TLR (e.g., TLR2 and/or TLR4). Thus, methodsdescribed herein include a nanoemulsion adjuvant composition (e.g.,composition comprising NE adjuvant optionally combined with one or moreimmunogens (e.g., Streptococcus (e.g., Streptococcus pneumoniae)antigens) that when administered to a subject, activates one or moreTLRs and stimulates an immune response (e.g., innate and/oradaptive/acquired immune response) in a subject. Such an adjuvant canactivate TLRs (e.g., TLR2 and/or TLR4) by, for example, interacting withTLRs (e.g., NE adjuvant binding to TLRs) or activating any downstreamcellular pathway that occurs upon binding of a ligand to a TLR. NEadjuvants described herein that activate TLRs can also enhance theavailability or accessibility of any endogenous or naturally occurringligand of TLRs. A NE adjuvant that activates one or more TLRs can altertranscription of genes, increase translation of mRNA or increase theactivity of proteins that are involved in mediating TLR cellularprocesses. For example, NE adjuvants described herein that activate oneor more TLRs (e.g., TLR2 and/or TLR4) can induce expression of one ormore cytokines (e.g., IL-8, IL-12p40, and/or IL-23)

As used herein, the term “immunity” refers to protection from disease(e.g., preventing or attenuating (e.g., suppression) of a sign, symptomor condition of the disease) upon exposure to a microorganism (e.g.,pathogen) capable of causing the disease. Immunity can be innate (e.g.,non-adaptive (e.g., non-acquired) immune responses that exist in theabsence of a previous exposure to an antigen) and/or acquired/adaptive(e.g., immune responses that are mediated by B and T cells following aprevious exposure to antigen (e.g., that exhibit increased specificityand reactivity to the antigen)).

As used herein, the terms “immunogen” and “antigen” refer to an agent(e.g., a microorganism (e.g., bacterium, virus or fungus) and/or portionor component thereof (e.g., a protein antigen)) that is capable ofeliciting an immune response in a subject. In preferred embodiments,immunogens elicit immunity against the immunogen (e.g., microorganism(e.g., pathogen or a pathogen product)) when administered in combinationwith a nanoemulsion of the present invention. As used herein, the termStreptococcus antigen refers to a component or product of a bacteria ofthe genus Streptococcus that elicits an immune response whenadministered to a subject.

As used herein, the term “pathogen product” refers to any component orproduct derived from a pathogen including, but not limited to,polypeptides, peptides, proteins, nucleic acids, membrane fractions, andpolysaccharides.

As used herein, the term “enhanced immunity” refers to an increase inthe level of adaptive and/or acquired immunity in a subject to a givenimmunogen (e.g., microorganism (e.g., pathogen)) followingadministration of a composition (e.g., composition for inducing animmune response of the present invention) relative to the level ofadaptive and/or acquired immunity in a subject that has not beenadministered the composition (e.g., composition for inducing an immuneresponse of the present invention).

As used herein, the terms “purified” or “to purify” refer to the removalof contaminants or undesired compounds from a sample or composition. Asused herein, the term “substantially purified” refers to the removal offrom about 70 to 90%, up to 100%, of the contaminants or undesiredcompounds from a sample or composition.

As used herein, the terms “administration” and “administering” refer tothe act of giving a composition of the present invention (e.g., acomposition for inducing an immune response (e.g., a compositioncomprising a nanoemulsion and an immunogen)) to a subject. Exemplaryroutes of administration to the human body include, but are not limitedto, through the eyes (ophthalmic), mouth (oral), skin (transdermal),nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, byinjection (e.g., intravenously, subcutaneously, intraperitoneally,etc.), topically, and the like.

As used herein, the terms “co-administration” and “co-administering”refer to the administration of at least two agent(s) (e.g., acomposition comprising a nanoemulsion and an immunogen and one or moreother agents—e.g., an adjuvant) or therapies to a subject. In someembodiments, the co-administration of two or more agents or therapies isconcurrent. In other embodiments, a first agent/therapy is administeredprior to a second agent/therapy. In some embodiments, co-administrationcan be via the same or different route of administration. Those of skillin the art understand that the formulations and/or routes ofadministration of the various agents or therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents or therapiesare co-administered, the respective agents or therapies are administeredat lower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents or therapies lowers the requisite dosageof a potentially harmful (e.g., toxic) agent(s), and/or whenco-administration of two or more agents results in sensitization of asubject to beneficial effects of one of the agents via co-administrationof the other agent. In other embodiments, co-administration ispreferable to elicit an immune response in a subject to two or moredifferent immunogens (e.g., microorganisms (e.g., pathogens)) at or nearthe same time (e.g., when a subject is unlikely to be available forsubsequent administration of a second, third, or more composition forinducing an immune response).

As used herein, the term “topically” refers to application of acompositions of the present invention (e.g., a composition comprising ananoemulsion and an immunogen) to the surface of the skin and/or mucosalcells and tissues (e.g., alveolar, buccal, lingual, masticatory, vaginalor nasal mucosa, and other tissues and cells which line hollow organs orbody cavities).

In some embodiments, the compositions of the present invention areadministered in the form of topical emulsions, injectable compositions,ingestible solutions, and the like. When the route is topical, the formmay be, for example, a spray (e.g., a nasal spray), a cream, or otherviscous solution (e.g., a composition comprising a nanoemulsion and animmunogen in polyethylene glycol).

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions (e.g., toxic, allergic orimmunological reactions) when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, and various types ofwetting agents (e.g., sodium lauryl sulfate), any and all solvents,dispersion media, coatings, sodium lauryl sulfate, isotonic andabsorption delaying agents, disintrigrants (e.g., potato starch orsodium starch glycolate), polyethyl glycol, and the like. Thecompositions also can include stabilizers and preservatives. Examples ofcarriers, stabilizers and adjuvants have been described and are known inthe art (See e.g., Martin, Remington's Pharmaceutical Sciences, 15thEd., Mack Publ. Co., Easton, Pa. (1975), incorporated herein byreference).

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acomposition of the present invention that is physiologically toleratedin the target subject. “Salts” of the compositions of the presentinvention may be derived from inorganic or organic acids and bases.Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compositions of the inventionand their pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,persulfate, phenylpropionate, picrate, pivalate, propionate, succinate,tartrate, thiocyanate, tosylate, undecanoate, and the like. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

For therapeutic use, salts of the compositions of the present inventionare contemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable composition.

As used herein, the term “at risk for disease” refers to a subject thatis predisposed to experiencing a particular disease. This predispositionmay be genetic (e.g., a particular genetic tendency to experience thedisease, such as heritable disorders), or due to other factors (e.g.,environmental conditions, exposures to detrimental compounds present inthe environment, etc.). Thus, it is not intended that the presentinvention be limited to any particular risk (e.g., a subject may be “atrisk for disease” simply by being exposed to and interacting with otherpeople), nor is it intended that the present invention be limited to anyparticular disease.

“Nasal application”, as used herein, means applied through the nose intothe nasal or sinus passages or both. The application may, for example,be done by drops, sprays, mists, coatings or mixtures thereof applied tothe nasal and sinus passages.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of immunogenic agents (e.g.,compositions comprising a nanoemulsion and an immunogen), such deliverysystems include systems that allow for the storage, transport, ordelivery of immunogenic agents and/or supporting materials (e.g.,written instructions for using the materials, etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant immunogenic agents (e.g., nanoemulsions) and/orsupporting materials. As used herein, the term “fragmented kit” refersto delivery systems comprising two or more separate containers that eachcontain a subportion of the total kit components. The containers may bedelivered to the intended recipient together or separately. For example,a first container may contain a composition comprising a nanoemulsionand an immunogen for a particular use, while a second container containsa second agent (e.g., an antibiotic or spray applicator). Indeed, anydelivery system comprising two or more separate containers that eachcontains a subportion of the total kit components are included in theterm “fragmented kit.” In contrast, a “combined kit” refers to adelivery system containing all of the components of an immunogenic agentneeded for a particular use in a single container (e.g., in a single boxhousing each of the desired components). The term “kit” includes bothfragmented and combined kits.

DETAILED DESCRIPTION OF THE INVENTION

Streptococcus pneumoniae is a Gram-positive bacterium responsible forconsiderable morbidity and mortality (particularly in the young andaged), causing invasive diseases such as pneumoniae, bacteraemia andmeningitis, and diseases associated with colonization, such as acuteOtitis media. The rate of pneumococcal pneumoniae in the US for personsover 60 years of age is estimated to be 3 to 8 per 100,000. In 20% ofcases this leads to bacteraemia, and other manifestations such asmeningitis, with a mortality rate close to 30% even with antibiotictreatment.

Pneumococcus is encapsulated with a chemically linked polysaccharidewhich confers serotype specificity. There are 90 known serotypes ofpneumococci, and the capsule is the principle virulence determinant forpneumococci, as the capsule not only protects the inner surface of thebacteria from complement, but is itself poorly immunogenic.Polysaccharides are T-independent antigens, and have been shown to notbe processed or presented on MHC molecules to interact with T-cells.They can however, stimulate the immune system through an alternatemechanism which involves cross-linking of surface receptors on B cells.It has been documents that protection against invasive pneumococcidisease is correlated most strongly with antibody specific for thecapsule, and the protection is serotype specific.

Streptococcus pneumoniae is the most common cause of invasive bacterialdisease and Otitis media in infants and young children. Likewise, theelderly mount poor responses to pneumococcal vaccines (See, e.g.,Roghmann et al., (1987), J. Gerontol. 42:265-270], hence the increasedincidence of bacterial pneumonia in this population (See, e.g., Vergheseand Berk, (1983) Medicine (Baltimore) 62:271-285).

Accordingly, the present invention provides methods and compositions forthe stimulation of immune responses. In particular, the presentinvention provides immunogenic nanoemulsion compositions and methods ofusing the same for the induction of immune responses (e.g., innateand/or adaptive immune responses (e.g., for generation of host immunityagainst a bacterial species of the genus Streptococcus (e.g.,Streptococcus pneumoniae))). Compositions and methods of the presentinvention find use in, among other things, clinical (e.g. therapeuticand preventative medicine (e.g., vaccination)) and researchapplications.

In some embodiments, the present invention provides nanoemulsionadjuvants and compositions comprising the same (e.g., vaccines) for thestimulation of immune responses (e.g., immunity) against a bacterialspecies of the genus Streptococcus (e.g., Streptococcus pneumoniae). Insome embodiments, the present invention provides nanoemulsion adjuvantcompositions that stimulate and/or elicit immune responses (e.g., innateimmune responses and/or adaptive/acquired immune responses) whenadministered to a subject (e.g., a human subject)). In some embodiments,the present invention provides nanoemulsion adjuvant compositionscomprising one or a plurality of Streptococcus (e.g., Streptococcuspneumoniae)) antigens (e.g., Streptococcus components and/or inactivatedStreptococcus). The present invention is not limited to any particularnanoemulsion or Streptococcus (e.g., Streptococcus pneumoniae)) antigen.Exemplary immunogenic compositions (e.g., vaccine compositions) andmethods of administering the compositions are described in more detailbelow.

In some embodiments, the present invention provides an immunogeniccomposition comprising a nanoemulsion and one or more Streptococcusantigens (e.g., Streptococcus pneumoniae antigens). In some embodiments,the present invention provides a method of inducing an immune responseto Streptococcus (e.g., Streptococcus pneumoniae) in a subjectcomprising: providing a subject and an immunogenic compositioncomprising a nanoemulsion and an immunogen, wherein the immunogencomprises a Streptococcus (e.g., Streptococcus pneumoniae) antigen andadministering the composition to the subject under conditions such thatthe subject generates a Streptococcus (e.g., Streptococcus pneumoniae)specific immune response. The present invention is not limited by theroute chosen for administration of a composition of the presentinvention. In some preferred embodiments, administering the immunogeniccomposition comprises contacting a mucosal surface of the subject withthe composition. In some embodiments, the mucosal surface comprisesnasal mucosa. In some embodiments, inducing an immune response inducesimmunity to Streptococcus (e.g., Streptococcus pneumoniae) in thesubject.

Experiments were conducted during development of embodiments of theinvention to determine if a composition comprising a nanoemulsion (NE)and Streptococcus pneumoniae antigen could be utilized to generate animmune response in a subject. Nasal immunization with a whole cellStreptococcus pneumoniae antigen (WCPAg) mixed with nanoemulsion wasperformed and shown to induce an IgG response in a host subject and theability to eradicate upper respiratory colonization of S. pneumoniae.

In particular, as described in Examples 1-4, experiments were conductedto determine whether S. pneumoniae mixed with nanoemulsion could producean immune response. CD-1 and C57/B6 mice were immunized with threeintranasal doses of WCPAg (1×10⁸ CFU or 1×10⁶ CFU) (See, e.g., Malley etal., (2001) Infect. Immun. 69, 4870-4873; Malley et al., (2004) Infect.Immun. 72, 4290-4292)) combined with various concentrations ofnanoemulsion. Serum antibody titers show that mixing of Staphylococcusantigen (WCPAg/W₈₀5EC) with nanoemulsion resulted in 10- to 20-foldincrease in immune response over the levels obtained withoutnanoemulsion, and was comparable with the standard intramuscularimmunization with alum adjuvant (See FIGS. 3A and 3B).

To test the protective effect of immunization mice were intranasallyinfected with S. pneumoniae. Analysis of bacteria recovered from thenasal washes 7 days post-colonization indicated protection againstpneumococcal colonization and decrease in colonization of the upperrespiratory tract in the mice immunized with 10⁸ CFU WCPAg plusnanoemulsion (See FIG. 4).

The efficacy of intranasal WCPAg/W₈₀5EC vaccine was evaluated at antigendoses of 7.5 μL and 0.1 μL in 1, 5, 10 and 20% nanoemulsion (NE). Theresults indicated that the doses of 7.5 μL, WCPAg in 1, 5, 10, and 20%NE elicited an immune response similar to that of the 7.5 μL, WCPAg+Alumgroup. Mucosal vaccination with 7.5 μL WCPAg in 1, 5, 10, and 20% NEproduced an immune response greater than 1×10⁵ mean IgG titers afterthree vaccinations. (See, e.g., Examples 2-4). Additionally, afterchallenging the mice with 1×10⁵ colony forming units (CFU) S.pneumoniae, there was a marked reduction in carriage in the 7.5 μL,WCPAg/NE and 7.5 μL+Alum groups versus control groups (See FIG. 4).Complete blood counts (white blood cells, neutrophils and monocytes)showed no abnormalities within any of the groups.

Accordingly, in some embodiments, the present invention provides thatadministration (e.g., nasal administration) of a composition comprisingnanoemulsion and S. pneumoniae antigen (e.g., whole cell S. pneumoniae)to a subject produces immunity toward S. pneumoniae in the subjectthereby protecting the subject against pneumococcal infection. In someembodiments, compositions and method of the present invention provideStreptococci (e.g., S. pneumoniae) specific protective immune responsesin a subject (e.g., similar to and/or greater than conventionalStreptococci (e.g., S. pneumoniae) vaccines (e.g., alum-adjuvantedvaccines))).

The present invention is not limited by the type of bacteria of thegenus Streptococci utilized in the immunogenic compositions and methodsof using the same of the invention. In some embodiments, the bacteria isa pathogen. In some embodiments, the pathogen is a Streptococcus speciesresponsible for strep throat, meningitis, bacterial pneumonia,endocarditis, erysipelas and/or necrotizing fasciitis. A variety ofStreptococcus species find use in the compositions and methods of theinvention including S. pneumoniae, S. mutans, S. mitis, S. sanguinis, S.salivarius, S. viridans, S. salivarius ssp. thermophilus, S.constellatus, S. pyogenes, S. agalactiae, S. zooepidemicus,Streptococcus bovis and Streptococcus equines, Streptococcus canis, aswell as former Group D Streptococci including S. faecalis, S. faecium,S. durans, and S. avium.

In some preferred embodiments, the bacteria of the genus Streptococci isS. pneumoniae. In some embodiments, an immunogenic compositioncomprising a nanoemulsion and S. pneumoniae antigen may compriseantigens (e.g., polysaccharide, protein, killed whole cells (e.g.,conjugated or non-conjugated antigens)), wherein the antigens arederived from multiple (e.g., at least 2, 3, 5, 7, 10, 15, 20, 30, 40,50, 60, 70, 80 or more) serotypes of S. pneumoniae. The number of S.pneumoniae antigens utilized can range from 10 different serotypes toabout 20 different serotypes. In another embodiment of the invention,the vaccine may comprise conjugated S. pneumoniae saccharides andunconjugated S. pneumoniae saccharides. For example, the invention maycomprise 10 conjugated serotypes and 10 unconjugated saccharides. Insome embodiments, an immunogenic composition comprising a nanoemulsionand S. pneumoniae antigen may comprise S. pneumoniae antigen (e.g.,whole cell, polysaccharide, protein, etc.) from every known and/orisolated serotype.

In some embodiments, an immunogenic composition comprising ananoemulsion and S. pneumoniae antigen comprises S. pneumoniae antigen(e.g., polysaccharide, protein, killed whole cells) selected from thefollowing serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, although it isappreciated other serotypes could be substituted depending on the age ofthe recipient receiving the vaccine and the geographical location wherethe vaccine will be administered. In some embodiments, a 10-valentvaccine refers to a composition comprising a nanoemulsion and S.pneumoniae antigen, wherein the S. pneumoniae antigen comprises antigen(e.g., polysaccharide, protein, killed whole cells) from 10 S.pneumoniae serotypes (e.g., serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19Fand 23F). In some embodiments, a 1-valent vaccine refers to acomposition comprising a nanoemulsion and S. pneumoniae antigen, whereinthe S. pneumoniae antigen comprises antigen (e.g., polysaccharide,protein, killed whole cells) from one S. pneumoniae serotype 3. In someembodiments, certain immunogenic compositions comprising a nanoemulsionand S. pneumoniae antigen comprise S. pneumoniae antigen (e.g.,polysaccharide, protein, killed whole cells) from a variety of serotypesassociated with pediatric infection (e.g., may comprise serotypes 6A and19A, or 6A and 22F, or 19A and 22F, or 6A and 15B, or 19A and 15B, or22F and 15B). In some embodiments, certain immunogenic compositionscomprising a nanoemulsion and S. pneumoniae antigen comprise S.pneumoniae antigen (e.g., polysaccharide, protein, killed whole cells)from a variety of serotypes associated with infection of the elderly(e.g., may comprise the serotypes 6A and 19A, or 6A and 22F, or 19A and22F, or 6A and 15B, or 19A and 15B, or 22F and 15B, supplemented withserotypes 19A and 22F, 8 and 12F, or 8 and 15B, or 8 and 19A, or 8 and22F, or 12F and 15B, or 12F and 19A, or 12F and 22F, or 15B and 19A, or15B and 22F).

In some embodiments, certain immunogenic compositions comprising ananoemulsion and S. pneumoniae antigen comprise S. pneumoniae antigen(e.g., polysaccharide, protein, killed whole cells) from a variety ofserotypes comprising serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A,11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

In some embodiments, an immunogenic composition comprising ananoemulsion and Streptococcus (e.g., Streptococcus pneumoniae) antigencomprises protein D (PD) from Haemophilus influenzae (see e.g. EP0594610). Haemophilus influenzae is a key causative organism of otitismedia (e.g., thereby protecting against Haemophilus influenzae relatedotitis media). In one embodiment, the vaccine composition comprisesprotein D. In one aspect, PD is present as a carrier protein. In anotheraspect, protein D is present in the vaccine composition as a freeprotein. In a further aspect, protein D is present both as a carrierprotein and as free protein. Protein D may be used as a full lengthprotein or as a fragment (See, e.g., WO0056360).

In some embodiments, an immunogenic composition comprising ananoemulsion and Streptococcus (e.g., Streptococcus pneumoniae) antigencomprises one, two or more different types of carrier protein (e.g.,that act as carriers for proteins, saccharides, etc.). For example, inone embodiment, two or more different saccharides or proteins may beconjugated to the same carrier protein, either to the same molecule ofcarrier protein or to different molecules of the same carrier protein.Carrier proteins may be TT, DT, CRM197, fragment C of TT, PhtD, PhtBE orPhtDE fusions (particularly those described in WO 01/98334 and WO03/54007), detoxified pneumolysin and protein D. In some embodiments, acarrier protein present in a composition comprising a nanoemulsion andStreptococcus (e.g., Streptococcus pneumoniae) antigen is a member ofthe polyhistidine triad family (Pht) proteins, fragments or fusionproteins thereof. The PhtA, PhtB, PhtD or PhtE proteins may have anamino acid sequence sharing 80%, 85%, 90%, 95%, 98%, 99% or 100%identity with a sequence disclosed in WO 00/37105 or WO 00/39299 (e.g.with amino acid sequence 1-838 or 21-838 of SEQ ID NO: 4 of WO 00/37105for PhtD). For example, fusion proteins are composed of full length orfragments of 2, 3 or 4 of PhtA, PhtB, PhtD, PhtE. Examples of fusionproteins are PhtA/B, PhtA/D, PhtA/E, PhtB/A, PhtB/D, PhtB/E. PhtD/A.PhtD/B, PhtD/E, PhtE/A, PhtE/B and PhtE/D, wherein the proteins arelinked with the first mentioned at the N-terminus (see for exampleWO01/98334). Carriers may comprise histidine triad motif(s) and/orcoiled coil regions. A histidine triad motif is the portion ofpolypeptide that has the sequence HxxHxH where H is histidine and x isan amino acid other than histidine. A coiled coil region is a regionpredicted by “Coils” algorithm Lupus, A et al (1991) Science 252;1162-1164.

Examples of carrier proteins which may be used in the present inventionare DT (Diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, DTCRM197 (a DT mutant) other DT point mutants, such as CRM176, CRM228, CRM45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45,CRM102, CRM 103 and CRM107 and other mutations described by Nicholls andYoule in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc,1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158to Gly and other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S.Pat. No. 4,950,740; mutation of at least one or more residues Lys 516,Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S.Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment disclosed inU.S. Pat. No. 5,843,711, pneumococcal pneumolysin (Kuo et al (1995)Infect Immun 63; 2706-13) including ply detoxified in some fashion forexample dPLY-GMBS (WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX,including PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins for examplePhtDE fusions, PhtBE fusions (WO 01/98334 and WO 03/54007), (Pht A-E aredescribed in more detail below) OMPC (meningococcal outer membraneprotein—usually extracted from N. meningitidis serogroup B-EP0372501),PorB (from N. meningitidis), PD (Haemophilus influenzae protein D—see,e.g., EP 0 594 610 B), or immunologically functional equivalentsthereof, synthetic peptides (EP0378881, EPO427347), heat shock proteins(WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EPO471177),cytokines, lymphokines, growth factors or hormones (WO 91/01146),artificial proteins comprising multiple human CD4+ T cell epitopes fromvarious pathogen derived antigens (Falugi et al (2001) Eur J Immunol 31;3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72;4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptakeproteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761).

Generation of Antibodies

An immunogenic composition comprising a nanoemulsion and Streptococcus(e.g., Streptococcus pneumoniae) antigen can be used to immunize amammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, toproduce polyclonal antibodies. If desired, a Streptococcus (e.g.,Streptococcus pneumoniae) antigen can be conjugated to a carrierprotein, such as bovine serum albumin, thyroglobulin, keyhole limpethemocyanin or other carrier described herein. Depending on the hostspecies, various adjuvants can be used to increase the immunologicalresponse. Such adjuvants include, but are not limited to, Freund'sadjuvant, mineral gels (e.g., aluminum hydroxide), and surface activesubstances (e.g. lysolecithin, pluronic polyols, polyanions, peptides,nanoemulsions described herein, keyhole limpet hemocyanin, anddinitrophenol). Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially useful.

Monoclonal antibodies that specifically bind to a Streptococcus (e.g.,Streptococcus pneumoniae) antigen can be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These techniques include, but are not limited to,the hybridoma technique, the human B cell hybridoma technique, and theEBV hybridoma technique (See, e.g., Kohler et al., Nature 256, 495 497,1985; Kozbor et al., J. Immunol. Methods 81, 3142, 1985; Cote et al.,Proc. Natl. Acad. Sci. 80, 2026 2030, 1983; Cole et al., Mol. Cell.Biol. 62, 109 120, 1984).

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (See, e.g., Morrison et al., Proc.Natl. Acad. Sci. 81, 68516855, 1984; Neuberger et al., Nature 312, 604608, 1984; Takeda et al., Nature 314, 452 454, 1985). Monoclonal andother antibodies also can be “humanized” to prevent a patient frommounting an immune response against the antibody when it is usedtherapeutically. Such antibodies may be sufficiently similar in sequenceto human antibodies to be used directly in therapy or may requirealteration of a few key residues. Sequence differences between rodentantibodies and human sequences can be minimized by replacing residueswhich differ from those in the human sequences by site directedmutagenesis of individual residues or by grating of entirecomplementarity determining regions.

Alternatively, humanized antibodies can be produced using recombinantmethods, as described below. Antibodies which specifically bind to aparticular antigen can contain antigen binding sites which are eitherpartially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies which specifically bind to a particular antigen.Antibodies with related specificity, but of distinct idiotypiccomposition, can be generated by chain shuffling from randomcombinatorial immunoglobin libraries (See, e.g., Burton, Proc. Natl.Acad. Sci. 88, 11120 23, 1991).

Single-chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(See, e.g., Thirion et al., 1996, Eur. J. Cancer Prey. 5, 507-11).Single-chain antibodies can be mono- or bispecific, and can be bivalentor tetravalent. Construction of tetravalent, bispecific single-chainantibodies is taught, for example, in Coloma & Morrison, 1997, Nat.Biotechnol. 15, 159-63. Construction of bivalent, bispecificsingle-chain antibodies is taught, for example, in Mallender & Voss,1994, J. Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence, as describedbelow. Alternatively, single-chain antibodies can be produced directlyusing, for example, filamentous phage technology (See, e.g., Verhaar etal., 1995, Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J.Immunol. Meth. 165, 81-91).

Antibodies which specifically bind to a particular antigen also can beproduced by inducing in vivo production in the lymphocyte population orby screening immunoglobulin libraries or panels of highly specificbinding reagents as disclosed in the literature (See, e.g., Orlandi etal., Proc. Natl. Acad. Sci. 86, 3833 3837, 1989; Winter et al., Nature349, 293 299, 1991).

Chimeric antibodies can be constructed as disclosed in WO 93/03151.Binding proteins which are derived from immunoglobulins and which aremultivalent and multispecific, such as the “diabodies” described in WO94/13804, also can be prepared. Antibodies can be purified by methodswell known in the art. For example, antibodies can be affinity purifiedby passage over a column to which the relevant antigen is bound. Thebound antibodies can then be eluted from the column using a buffer witha high salt concentration.

Nanoemulsions

The present invention is not limited by the type of nanoemulsionadjuvant utilized (e.g., for respiratory administration). Indeed, avariety of nanoemulsion adjuvants are contemplated to be useful in thepresent invention.

For example, in some embodiments, a nanoemulsion comprises (i) anaqueous phase; (ii) an oil phase; and at least one additional compound.In some embodiments of the present invention, these additional compoundsare admixed into either the aqueous or oil phases of the composition. Inother embodiments, these additional compounds are admixed into acomposition of previously emulsified oil and aqueous phases. In certainof these embodiments, one or more additional compounds are admixed intoan existing emulsion composition immediately prior to its use. In otherembodiments, one or more additional compounds are admixed into anexisting emulsion composition prior to the compositions immediate use.

Additional compounds suitable for use in a nanoemulsion of the presentinvention include, but are not limited to, one or more organic, and moreparticularly, organic phosphate based solvents, surfactants anddetergents, cationic halogen containing compounds, germinationenhancers, interaction enhancers, food additives (e.g., flavorings,sweeteners, bulking agents, and the like) and pharmaceuticallyacceptable compounds (e.g., carriers). Certain exemplary embodiments ofthe various compounds contemplated for use in the compositions of thepresent invention are presented below. Unless described otherwise,nanoemulsions are described in undiluted form.

Nanoemulsion adjuvant compositions of the present invention are notlimited to any particular nanoemulsion. Any number of suitablenanoemulsion compositions may be utilized in the vaccine compositions ofthe present invention, including, but not limited to, those disclosed inHamouda et al., J. Infect Dis., 180:1939 (1999); Hamouda and Baker, J.Appl. Microbiol., 89:397 (2000); and Donovan et al., Antivir. Chem.Chemother., 11:41 (2000). Preferred nanoemulsions of the presentinvention are those that are non-toxic to animals. In preferredembodiments, nanoemulsions utilized in the methods of the presentinvention are stable, and do not decompose even after long storageperiods (e.g., one or more years). Additionally, preferred emulsionsmaintain stability even after exposure to high temperature and freezing.This is especially useful if they are to be applied in extremeconditions (e.g., extreme heat or cold). Some embodiments of the presentinvention employ an oil phase containing ethanol.

For example, in some embodiments, the emulsions of the present inventioncontain (i) an aqueous phase and (ii) an oil phase containing ethanol asthe organic solvent and optionally a germination enhancer, and (iii)TYLOXAPOL as the surfactant (preferably 2-5%, more preferably 3%). Thisformulation is highly efficacious for inactivation of pathogens and isalso non-irritating and non-toxic to mammalian subjects (e.g., and thuscan be used for administration to a mucosal surface).

In some other embodiments, the emulsions of the present inventioncomprise a first emulsion emulsified within a second emulsion, wherein(a) the first emulsion comprises (i) an aqueous phase; and (ii) an oilphase comprising an oil and an organic solvent; and (iii) a surfactant;and (b) the second emulsion comprises (i) an aqueous phase; and (ii) anoil phase comprising an oil and a cationic containing compound; and(iii) a surfactant.

Exemplary Formulations

The following description provides a number of exemplary emulsionsincluding formulations for compositions BCTP and X₈W₆₀PC. BCTP comprisesa water-in oil nanoemulsion, in which the oil phase was made fromsoybean oil, tri-n-butyl phosphate, and TRITON X-100 in 80% water.X₈W₆₀PC comprises a mixture of equal volumes of BCTP with W₈₀8P. W₈₀8Pis a liposome-like compound made of glycerol monostearate, refined oyasterols (e.g., GENEROL sterols), TWEEN 60, soybean oil, a cationic ionhalogen-containing CPC and peppermint oil. The GENEROL family are agroup of a polyethoxylated soya sterols (Henkel Corporation, Ambler,Pa.). Exemplary emulsion formulations useful in the present inventionare provided in Table 1. These particular formulations may be found inU.S. Pat. Nos. 5,700,679 (NN); 5,618,840; 5,549,901 (W₈₀8P); and5,547,677, each of which is hereby incorporated by reference in theirentireties. Certain other emulsion formulations are presented U.S.patent application Ser. No. 10/669,865, hereby incorporated by referencein its entirety.

The X₈W₆₀PC emulsion is manufactured by first making the W₈₀8P emulsionand BCTP emulsions separately. A mixture of these two emulsions is thenre-emulsified to produce a fresh emulsion composition termed X₈W₆₀PC.Methods of producing such emulsions are described in U.S. Pat. Nos.5,103,497 and 4,895,452 (each of which is herein incorporated byreference in their entireties).

TABLE 1 Water to Oil Phase Ratio Oil Phase Formula (Vol/Vol) BCTP 1 vol.Tri(N-butyl)phosphate   4:1 1 vol. TRITON X-100 8 vol. Soybean oil NN86.5 g Glycerol monooleate   3:1 60.1 ml Nonoxynol-9 24.2 g GENEROL 1223.27 g Cetylpyridinium chloride 554 g Soybean oil W₈₀8P 86.5 g Glycerolmonooleate 3.2:1 21.2 g Polysorbate 60 24.2 g GENEROL 122 3.27 gCetylpyddinium chloride 4 ml Peppermint oil 554 g Soybean oil SS 86.5 gGlycerol monooleate 3.2:1 21.2 g Polysorbate 60 (1% bismuth in water)24.2 g GENEROL 122 3.27 g Cetylpyridinium chloride 554 g Soybean oil

The compositions listed above are only exemplary and those of skill inthe art will be able to alter the amounts of the components to arrive ata nanoemulsion composition suitable for the purposes of the presentinvention. Those skilled in the art will understand that the ratio ofoil phase to water as well as the individual oil carrier, surfactant CPCand organic phosphate buffer, components of each composition may vary.

Although certain compositions comprising BCTP have a water to oil ratioof 4:1, it is understood that the BCTP may be formulated to have more orless of a water phase. For example, in some embodiments, there is 3, 4,5, 6, 7, 8, 9, 10, or more parts of the water phase to each part of theoil phase. The same holds true for the W₈₀8P formulation. Similarly, theratio of Tri (N-butyl) phosphate: TRITON X-100: soybean oil also may bevaried.

Although Table 1 lists specific amounts of glycerol monooleate,polysorbate 60, GENEROL 122, cetylpyridinium chloride, and carrier oilfor W₈₀8P, these are merely exemplary. An emulsion that has theproperties of W₈₀8P may be formulated that has different concentrationsof each of these components or indeed different components that willfulfill the same function. For example, the emulsion may have betweenabout 80 to about 100 g of glycerol monooleate in the initial oil phase.In other embodiments, the emulsion may have between about 15 to about 30g polysorbate 60 in the initial oil phase. In yet another embodiment thecomposition may comprise between about 20 to about 30 g of a GENEROLsterol, in the initial oil phase.

Individual components of nanoemulsions (e.g. in an immunogeniccomposition of the present invention) can function both to inactivate apathogen as well as to contribute to the non-toxicity of the emulsions.For example, the active component in BCTP, TRITON-X100, shows lessability to inactivate a virus at concentrations equivalent to 11% BCTP.Adding the oil phase to the detergent and solvent markedly reduces thetoxicity of these agents in tissue culture at the same concentrations.While not being bound to any theory (an understanding of the mechanismis not necessary to practice the present invention, and the presentinvention is not limited to any particular mechanism), it is suggestedthat the nanoemulsion enhances the interaction of its components withthe pathogens thereby facilitating the inactivation of the pathogen andreducing the toxicity of the individual components. Furthermore, whenall the components of BCTP are combined in one composition but are notin a nanoemulsion structure, the mixture is not as effective atinactivating a pathogen as when the components are in a nanoemulsionstructure.

Numerous additional embodiments presented in classes of formulationswith like compositions are presented below. The following compositionsrecite various ratios and mixtures of active components. One skilled inthe art will appreciate that the below recited formulation are exemplaryand that additional formulations comprising similar percent ranges ofthe recited components are within the scope of the present invention.

In certain embodiments of the present invention, a nanoemulsioncomprises from about 3 to 8 vol. % of TYLOXAPOL, about 8 vol. % ofethanol, about 1 vol. % of cetylpyridinium chloride (CPC), about 60 to70 vol. % oil (e.g., soybean oil), about 15 to 25 vol. % of aqueousphase (e.g., DiH₂O or PBS), and in some formulations less than about 1vol. % of 1N NaOH. Some of these embodiments comprise PBS. It iscontemplated that the addition of 1N NaOH and/or PBS in some of theseembodiments, allows the user to advantageously control the pH of theformulations, such that pH ranges from about 7.0 to about 9.0, and morepreferably from about 7.1 to 8.5 are achieved. For example, oneembodiment of the present invention comprises about 3 vol. % ofTYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 64vol. % of soybean oil, and about 24 vol. % of DiH₂O (designated hereinas Y3EC). Another similar embodiment comprises about 3.5 vol. % ofTYLOXAPOL, about 8 vol. % of ethanol, and about 1 vol. % of CPC, about64 vol. % of soybean oil, and about 23.5 vol. % of DiH₂O (designatedherein as Y3.5EC). Yet another embodiment comprises about 3 vol. % ofTYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 0.067vol. % of 1N NaOH, such that the pH of the formulation is about 7.1,about 64 vol. % of soybean oil, and about 23.93 vol. % of DiH₂O(designated herein as Y3EC pH 7.1). Still another embodiment comprisesabout 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. %of CPC, about 0.67 vol. % of 1N NaOH, such that the pH of theformulation is about 8.5, and about 64 vol. % of soybean oil, and about23.33 vol. % of DiH₂O (designated herein as Y3EC pH 8.5). Anothersimilar embodiment comprises about 4% TYLOXAPOL, about 8 vol. % ethanol,about 1% CPC, and about 64 vol. % of soybean oil, and about 23 vol. % ofDiH₂O (designated herein as Y4EC). In still another embodiment theformulation comprises about 8% TYLOXAPOL, about 8% ethanol, about 1 vol.% of CPC, and about 64 vol. % of soybean oil, and about 19 vol. % ofDiH₂O (designated herein as Y8EC). A further embodiment comprises about8 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC,about 64 vol. % of soybean oil, and about 19 vol. % of 1×PBS (designatedherein as Y8EC PBS).

In some embodiments of the present invention, a nanoemulsion comprisesabout 8 vol. % of ethanol, and about 1 vol. % of CPC, and about 64 vol.% of oil (e.g., soybean oil), and about 27 vol. % of aqueous phase(e.g., DiH₂O or PBS) (designated herein as EC).

In some embodiments, a nanoemulsion comprises from about 8 vol. % ofsodium dodecyl sulfate (SDS), about 8 vol. % of tributyl phosphate(TBP), and about 64 vol. % of oil (e.g., soybean oil), and about 20 vol.% of aqueous phase (e.g., DiH₂O or PBS) (designated herein as S8P).

In some embodiments, a nanoemulsion comprises from about 1 to 2 vol. %of TRITON X-100, from about 1 to 2 vol. % of TYLOXAPOL, from about 7 to8 vol. % of ethanol, about 1 vol. % of cetylpyridinium chloride (CPC),about 64 to 57.6 vol. % of oil (e.g., soybean oil), and about 23 vol. %of aqueous phase (e.g., DiH₂O or PBS). Additionally, some of theseformulations further comprise about 5 mM of L-alanine/Inosine, and about10 mM ammonium chloride. Some of these formulations comprise PBS. It iscontemplated that the addition of PBS in some of these embodiments,allows the user to advantageously control the pH of the formulations.For example, one embodiment of the present invention comprises about 2vol. % of TRITON X-100, about 2 vol. % of TYLOXAPOL, about 8 vol. % ofethanol, about 1 vol. % CPC, about 64 vol. % of soybean oil, and about23 vol. % of aqueous phase DiH₂O. In another embodiment the formulationcomprises about 1.8 vol. % of TRITON X-100, about 1.8 vol. % ofTYLOXAPOL, about 7.2 vol. % of ethanol, about 0.9 vol. % of CPC, about 5mM L-alanine/Inosine, and about 10 mM ammonium chloride, about 57.6 vol.% of soybean oil, and the remainder of 1×PBS (designated herein as 90%X2Y2EC/GE).

In alternative embodiments, a nanoemulsion comprises from about 5 vol. %of TWEEN 80, from about 8 vol. % of ethanol, from about 1 vol. % of CPC,about 64 vol. % of oil (e.g., soybean oil), and about 22 vol. % of DiH₂O(designated herein as W₈₀5EC). In yet another alternative embodiment, ananoemulsion comprises from about 5 vol. % of TWEEN 80, from about 8vol. % of ethanol, about 64 vol. % of oil (e.g., soybean oil), and about23 vol. % of DiH₂O (designated herein as W₈₀5E).

In some embodiments, the present invention provides a nanoemulsioncomprising from about 5 vol. % of Poloxamer-407, from about 8 vol. % ofethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g.,soybean oil), and about 22 vol. % of DiH₂O (designated herein asP₄₀₇5EC). Although an understanding of the mechanism is not necessary topractice the present invention, and the present invention is not limitedto any particular mechanism, in some embodiments, a nanoemulsioncomprising Poloxamer-407 does not elicit and/or augment immune responses(e.g., in the lung) in a subject. In some embodiments, various dilutionsof a nanoemulsion provided herein (e.g., P₄₀₇5EC) can be utilized totreat (e.g., kill and/or inhibit growth of) bacteria. In someembodiments, undiluted nanoemulsion is utilized. In some embodiments,P₄₀₇5EC is diluted (e.g., in serial, two fold dilutions) to obtain adesired concentration of one of the constituents of the nanoemulsion(e.g., CPC).

In still other embodiments of the present invention, a nanoemulsioncomprises from about 5 vol. % of TWEEN 20, from about 8 vol. % ofethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g.,soybean oil), and about 22 vol. % of DiH₂O (designated herein asW₂₀5EC).

In still other embodiments of the present invention, a nanoemulsioncomprises from about 2 to 8 vol. % of TRITON X-100, about 8 vol. % ofethanol, about 1 vol. % of CPC, about 60 to 70 vol. % of oil (e.g.,soybean, or olive oil), and about 15 to 25 vol. % of aqueous phase(e.g., DiH₂O or PBS). For example, the present invention contemplatesformulations comprising about 2 vol. % of TRITON X-100, about 8 vol. %of ethanol, about 64 vol. % of soybean oil, and about 26 vol. % of DiH₂O(designated herein as X2E). In other similar embodiments, a nanoemulsioncomprises about 3 vol. % of TRITON X-100, about 8 vol. % of ethanol,about 64 vol. % of soybean oil, and about 25 vol. % of DiH₂O (designatedherein as X3E). In still further embodiments, the formulations compriseabout 4 vol. % Triton of X-100, about 8 vol. % of ethanol, about 64 vol.% of soybean oil, and about 24 vol. % of DiH₂O (designated herein asX4E). In yet other embodiments, a nanoemulsion comprises about 5 vol. %of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybeanoil, and about 23 vol. % of DiH₂O (designated herein as X5E). In someembodiments, a nanoemulsion comprises about 6 vol. % of TRITON X-100,about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 22vol. % of DiH₂O (designated herein as X6E). In still further embodimentsof the present invention, a nanoemulsion comprises about 8 vol. % ofTRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil,and about 20 vol. % of DiH₂O (designated herein as X8E). In stillfurther embodiments, a nanoemulsion comprises about 8 vol. % of TRITONX-100, about 8 vol. % of ethanol, about 64 vol. % of olive oil, andabout 20 vol. % of DiH₂O (designated herein as X8E 0). In yet anotherembodiment, a nanoemulsion comprises 8 vol. % of TRITON X-100, about 8vol. % ethanol, about 1 vol. % CPC, about 64 vol. % of soybean oil, andabout 19 vol. % of DiH₂O (designated herein as X8EC).

In alternative embodiments of the present invention, a nanoemulsioncomprises from about 1 to 2 vol. % of TRITON X-100, from about 1 to 2vol. % of TYLOXAPOL, from about 6 to 8 vol. % TBP, from about 0.5 to 1.0vol. % of CPC, from about 60 to 70 vol. % of oil (e.g., soybean), andabout 1 to 35 vol. % of aqueous phase (e.g., DiH₂O or PBS).Additionally, certain of these nanoemulsions may comprise from about 1to 5 vol. % of trypticase soy broth, from about 0.5 to 1.5 vol. % ofyeast extract, about 5 mM L-alanine/Inosine, about 10 mM ammoniumchloride, and from about 20-40 vol. % of liquid baby formula. In someembodiments comprising liquid baby formula, the formula comprises acasein hydrolysate (e.g., Neutramigen, or Progestimil, and the like). Insome of these embodiments, a nanoemulsion further comprises from about0.1 to 1.0 vol. % of sodium thiosulfate, and from about 0.1 to 1.0 vol.% of sodium citrate. Other similar embodiments comprising these basiccomponents employ phosphate buffered saline (PBS) as the aqueous phase.For example, one embodiment comprises about 2 vol. % of TRITON X-100,about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1 vol. % of CPC,about 64 vol. % of soybean oil, and about 23 vol. % of DiH₂O (designatedherein as X2Y2EC). In still other embodiments, the inventive formulationcomprises about 2 vol. % of TRITON X-100, about 2 vol. % TYLOXAPOL,about 8 vol. % TBP, about 1 vol. % of CPC, about 0.9 vol. % of sodiumthiosulfate, about 0.1 vol. % of sodium citrate, about 64 vol. % ofsoybean oil, and about 22 vol. % of DiH₂O (designated herein as X2Y2PCSTS1). In another similar embodiment, a nanoemulsion comprises about 1.7vol. % TRITON X-100, about 1.7 vol. % TYLOXAPOL, about 6.8 vol. % TBP,about 0.85% CPC, about 29.2% NEUTRAMIGEN, about 54.4 vol. % of soybeanoil, and about 4.9 vol. % of DiH₂O (designated herein as 85%X2Y2PC/baby). In yet another embodiment of the present invention, ananoemulsion comprises about 1.8 vol. % of TRITON X-100, about 1.8 vol.% of TYLOXAPOL, about 7.2 vol. % of TBP, about 0.9 vol. % of CPC, about5 mM L-alanine/Inosine, about 10 mM ammonium chloride, about 57.6 vol. %of soybean oil, and the remainder vol. % of 0.1x PBS (designated hereinas 90% X2Y2 PC/GE). In still another embodiment, a nanoemulsioncomprises about 1.8 vol. % of TRITON X-100, about 1.8 vol. % ofTYLOXAPOL, about 7.2 vol. % TBP, about 0.9 vol. % of CPC, and about 3vol. % trypticase soy broth, about 57.6 vol. % of soybean oil, and about27.7 vol. % of DiH₂O (designated herein as 90% X2Y2PC/TSB). In anotherembodiment of the present invention, a nanoemulsion comprises about 1.8vol. % TRITON X-100, about 1.8 vol. % TYLOXAPOL, about 7.2 vol. % TBP,about 0.9 vol. % CPC, about 1 vol. % yeast extract, about 57.6 vol. % ofsoybean oil, and about 29.7 vol. % of DiH₂O (designated herein as 90%X2Y2PC/YE).

In some embodiments of the present invention, a nanoemulsion comprisesabout 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1 vol. %of CPC, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), andabout 15 to 30 vol. % of aqueous phase (e.g., DiH₂O or PBS). In aparticular embodiment of the present invention, a nanoemulsion comprisesabout 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1 vol. %of CPC, about 64 vol. % of soybean, and about 24 vol. % of DiH₂O(designated herein as Y3PC).

In some embodiments of the present invention, a nanoemulsion comprisesfrom about 4 to 8 vol. % of TRITON X-100, from about 5 to 8 vol. % ofTBP, about 30 to 70 vol. % of oil (e.g., soybean or olive oil), andabout 0 to 30 vol. % of aqueous phase (e.g., DiH₂O or PBS).Additionally, certain of these embodiments further comprise about 1 vol.% of CPC, about 1 vol. % of benzalkonium chloride, about 1 vol. %cetylyridinium bromide, about 1 vol. % cetyldimethyletylammoniumbromide, 500 μM EDTA, about 10 mM ammonium chloride, about 5 mM Inosine,and about 5 mM L-alanine. For example, in a certain preferredembodiment, a nanoemulsion comprises about 8 vol. % of TRITON X-100,about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 20 vol.% of DiH₂O (designated herein as X8P). In another embodiment of thepresent invention, a nanoemulsion comprises about 8 vol. % of TRITONX-100, about 8 vol. % of TBP, about 1% of CPC, about 64 vol. % ofsoybean oil, and about 19 vol. % of DiH₂O (designated herein as X8PC).In still another embodiment, a nanoemulsion comprises about 8 vol. %TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 50vol. % of soybean oil, and about 33 vol. % of DiH₂O (designated hereinas ATB-X1001). In yet another embodiment, the formulations compriseabout 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol. % ofCPC, about 50 vol. % of soybean oil, and about 32 vol. % of DiH₂O(designated herein as ATB-X002). In some embodiments, a nanoemulsioncomprises about 4 vol. % TRITON X-100, about 4 vol. % of TBP, about 0.5vol. % of CPC, about 32 vol. % of soybean oil, and about 59.5 vol. % ofDiH₂O (designated herein as 50% X8PC). In some embodiments, ananoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol. % ofTBP, about 0.5 vol. % CPC, about 64 vol. % of soybean oil, and about19.5 vol. % of DiH₂O (designated herein as X8PC_(1/2)). In someembodiments of the present invention, a nanoemulsion comprises about 8vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol. % of CPC,about 64 vol. % of soybean oil, and about 18 vol. % of DiH₂O (designatedherein as X8PC2). In other embodiments, a nanoemulsion comprises about 8vol. % of TRITON X-100, about 8% of TBP, about 1% of benzalkoniumchloride, about 50 vol. % of soybean oil, and about 33 vol. % of DiH₂O(designated herein as X8P BC). In an alternative embodiment of thepresent invention, a nanoemulsion comprises about 8 vol. % of TRITONX-100, about 8 vol. % of TBP, about 1 vol. % of cetylyridinium bromide,about 50 vol. % of soybean oil, and about 33 vol. % of DiH₂O (designatedherein as X8P CPB). In another exemplary embodiment of the presentinvention, a nanoemulsion comprises about 8 vol. % of TRITON X-100,about 8 vol. % of TBP, about 1 vol. % of cetyldimethyletylammoniumbromide, about 50 vol. % of soybean oil, and about 33 vol. % of DiH₂O(designated herein as X8P CTAB). In still further embodiments, ananoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol. % ofTBP, about 1 vol. % of CPC, about 500 μM EDTA, about 64 vol. % ofsoybean oil, and about 15.8 vol. % DiH₂O (designated herein as X8PCEDTA). In some embodiments, a nanoemulsion comprises 8 vol. % of TRITONX-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 10 mMammonium chloride, about 5 mM Inosine, about 5 mM L-alanine, about 64vol. % of soybean oil, and about 19 vol. % of DiH₂O or PBS (designatedherein as X8PC GE_(1x)). In another embodiment of the present invention,a nanoemulsion comprises about 5 vol. % of TRITON X-100, about 5% ofTBP, about 1 vol. % of CPC, about 40 vol. % of soybean oil, and about 49vol. % of DiH₂O (designated herein as X5P₅C).

In some embodiments of the present invention, a nanoemulsion comprisesabout 2 vol. % TRITON X-100, about 6 vol. % TYLOXAPOL, about 8 vol. %ethanol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH₂O(designated herein as X2Y6E).

In an additional embodiment of the present invention, a nanoemulsioncomprises about 8 vol. % of TRITON X-100, and about 8 vol. % ofglycerol, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), andabout 15 to 25 vol. % of aqueous phase (e.g., DiH₂O or PBS). Certainnanoemulsion compositions (e.g., used to generate an immune response(e.g., for use as a vaccine) comprise about 1 vol. % L-ascorbic acid.For example, one particular embodiment comprises about 8 vol. % ofTRITON X-100, about 8 vol. % of glycerol, about 64 vol. % of soybeanoil, and about 20 vol. % of DiH₂O (designated herein as X8G). In stillanother embodiment, a nanoemulsion comprises about 8 vol. % of TRITONX-100, about 8 vol. % of glycerol, about 1 vol. % of L-ascorbic acid,about 64 vol. % of soybean oil, and about 19 vol. % of DiH₂O (designatedherein as X8GV_(c)).

In still further embodiments, a nanoemulsion comprises about 8 vol. % ofTRITON X-100, from about 0.5 to 0.8 vol. % of TWEEN 60, from about 0.5to 2.0 vol. % of CPC, about 8 vol. % of TBP, about 60 to 70 vol. % ofoil (e.g., soybean or olive oil), and about 15 to 25 vol. % of aqueousphase (e.g., DiH₂O or PBS). For example, in one particular embodiment ananoemulsion comprises about 8 vol. % of TRITON X-100, about 0.70 vol. %of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol.% of soybean oil, and about 18.3 vol. % of DiH₂O (designated herein asX8W60PC₁). In some embodiments, a nanoemulsion comprises about 8 vol. %of TRITON X-100, about 0.71 vol. % of TWEEN 60, about 1 vol. % of CPC,about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 18.29vol. % of DiH₂O (designated herein as W60_(0.7)X8PC). In yet otherembodiments, a nanoemulsion comprises from about 8 vol. % of TRITONX-100, about 0.7 vol. % of TWEEN 60, about 0.5 vol. % of CPC, about 8vol. % of TBP, about 64 to 70 vol. % of soybean oil, and about 18.8 vol.% of DiH₂O (designated herein as X8W60PC₂). In still other embodiments,a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 0.71 vol.% of TWEEN 60, about 2 vol. % of CPC, about 8 vol. % of TBP, about 64vol. % of soybean oil, and about 17.3 vol. % of DiH₂O. In anotherembodiment of the present invention, a nanoemulsion comprises about 0.71vol. % of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % of TBP, about64 vol. % of soybean oil, and about 25.29 vol. % of DiH₂O (designatedherein as W60_(0.7)PC).

In another embodiment of the present invention, a nanoemulsion comprisesabout 2 vol. % of dioctyl sulfosuccinate, either about 8 vol. % ofglycerol, or about 8 vol. % TBP, in addition to, about 60 to 70 vol. %of oil (e.g., soybean or olive oil), and about 20 to 30 vol. % ofaqueous phase (e.g., DiH₂O or PBS). For example, in some embodiments, ananoemulsion comprises about 2 vol. % of dioctyl sulfosuccinate, about 8vol. % of glycerol, about 64 vol. % of soybean oil, and about 26 vol. %of DiH₂O (designated herein as D2G). In another related embodiment, ananoemulsion comprises about 2 vol. % of dioctyl sulfosuccinate, andabout 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 26 vol.% of DiH₂O (designated herein as D2P).

In still other embodiments of the present invention, a nanoemulsioncomprises about 8 to 10 vol. % of glycerol, and about 1 to 10 vol. % ofCPC, about 50 to 70 vol. % of oil (e.g., soybean or olive oil), andabout 15 to 30 vol. % of aqueous phase (e.g., DiH₂O or PBS).Additionally, in certain of these embodiments, a nanoemulsion furthercomprises about 1 vol. % of L-ascorbic acid. For example, in someembodiments, a nanoemulsion comprises about 8 vol. % of glycerol, about1 vol. % of CPC, about 64 vol. % of soybean oil, and about 27 vol. % ofDiH₂O (designated herein as GC). In some embodiments, a nanoemulsioncomprises about 10 vol. % of glycerol, about 10 vol. % of CPC, about 60vol. % of soybean oil, and about 20 vol. % of DiH₂O (designated hereinas GC10). In still another embodiment of the present invention, ananoemulsion comprises about 10 vol. % of glycerol, about 1 vol. % ofCPC, about 1 vol. % of L-ascorbic acid, about 64 vol. % of soybean oroil, and about 24 vol. % of DiH₂O (designated herein as GCV_(c)).

In some embodiments of the present invention, a nanoemulsion comprisesabout 8 to 10 vol. % of glycerol, about 8 to 10 vol. % of SDS, about 50to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30vol. % of aqueous phase (e.g., DiH₂O or PBS). Additionally, in certainof these embodiments, a nanoemulsion further comprise about 1 vol. % oflecithin, and about 1 vol. % of p-Hydroxybenzoic acid methyl ester.Exemplary embodiments of such formulations comprise about 8 vol. % SDS,8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol.% of DiH₂O (designated herein as S8G). A related formulation comprisesabout 8 vol. % of glycerol, about 8 vol. % of SDS, about 1 vol. % oflecithin, about 1 vol. % of p-Hydroxybenzoic acid methyl ester, about 64vol. % of soybean oil, and about 18 vol. % of DiH₂O (designated hereinas S8GL1B1).

In yet another embodiment of the present invention, a nanoemulsioncomprises about 4 vol. % of TWEEN 80, about 4 vol. % of TYLOXAPOL, about1 vol. % of CPC, about 8 vol. % of ethanol, about 64 vol. % of soybeanoil, and about 19 vol. % of DiH₂O (designated herein as W₈₀4Y4EC).

In some embodiments of the present invention, a nanoemulsion comprisesabout 0.01 vol. % of CPC, about 0.08 vol. % of TYLOXAPOL, about 10 vol.% of ethanol, about 70 vol. % of soybean oil, and about 19.91 vol. % ofDiH₂O (designated herein as Y.08EC.01).

In yet another embodiment of the present invention, a nanoemulsioncomprises about 8 vol. % of sodium lauryl sulfate, and about 8 vol. % ofglycerol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH₂O(designated herein as SLS8G).

The specific formulations described above are simply examples toillustrate the variety of nanoemulsion adjuvants that find use in thepresent invention. The present invention contemplates that manyvariations of the above formulations, as well as additionalnanoemulsions, find use in the methods of the present invention.Candidate emulsions can be easily tested to determine if they aresuitable. First, the desired ingredients are prepared using the methodsdescribed herein, to determine if an emulsion can be formed. If anemulsion cannot be formed, the candidate is rejected. For example, acandidate composition made of 4.5% sodium thiosulfate, 0.5% sodiumcitrate, 10% n-butanol, 64% soybean oil, and 21% DiH₂O does not form anemulsion.

Second, the candidate emulsion should form a stable emulsion. Anemulsion is stable if it remains in emulsion form for a sufficientperiod to allow its intended use (e.g., to generate an immune responsein a subject). For example, for emulsions that are to be stored,shipped, etc., it may be desired that the composition remain in emulsionform for months to years. Typical emulsions that are relativelyunstable, will lose their form within a day. For example, a candidatecomposition made of 8% 1-butanol, 5% TWEEN 10, 1% CPC, 64% soybean oil,and 22% DiH₂O does not form a stable emulsion. Nanoemulsions that havebeen shown to be stable include, but are not limited to, 8 vol. % ofTRITON X-100, about 8 vol. % of TBP, about 64 vol. % of soybean oil, andabout 20 vol. % of DiH₂O (designated herein as X8P); 5 vol. % of TWEEN20, from about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64vol. % of oil (e.g., soybean oil), and about 22 vol. % of DiH₂O(designated herein as W₂₀5EC); 0.08% Triton X-100, 0.08% Glycerol, 0.01%Cetylpyridinium Chloride, 99% Butter, and 0.83% diH₂O (designated hereinas 1% X8GC Butter); 0.8% Triton X-100, 0.8% Glycerol, 0.1%Cetylpyridinium Chloride, 6.4% Soybean Oil, 1.9% diH₂O, and 90% Butter(designated herein as 10% X8GC Butter); 2% W₂₀5EC, 1% Natrosol 250L NF,and 97% diH₂O (designated herein as 2% W₂₀5EC L GEL); 1% CetylpyridiniumChloride, 5% TWEEN 20, 8% Ethanol, 64% 70 Viscosity Mineral Oil, and 22%diH₂O (designated herein as W₂₀5EC70 Mineral Oil); 1% CetylpyridiniumChloride, 5% TWEEN 20, 8% Ethanol, 64% 350 Viscosity Mineral Oil, and22% diH₂O (designated herein as W₂₀5EC350 Mineral Oil). In someembodiments, nanoemulsions of the present invention are stable for overa week, over a month, or over a year.

Third, the candidate emulsion should have efficacy for its intended use.For example, a nanoemuslion should inactivate (e.g., kill or inhibitgrowth of) a pathogen to a desired level (e.g., 1 log, 2 log, 3 log, 4log, . . . reduction). Using the methods described herein, one iscapable of determining the suitability of a particular candidateemulsion against the desired pathogen. Generally, this involves exposingthe pathogen to the emulsion for one or more time periods in aside-by-side experiment with the appropriate control samples (e.g., anegative control such as water) and determining if, and to what degree,the emulsion inactivates (e.g., kills and/or neutralizes) themicroorganism. For example, a candidate composition made of 1% ammoniumchloride, 5% TWEEN 20, 8% ethanol, 64% soybean oil, and 22% DiH₂O wasshown not to be an effective emulsion. The following candidate emulsionswere shown to be effective using the methods described herein: 5% TWEEN20, 5% Cetylpyridinium Chloride, 10% Glycerol, 60% Soybean Oil, and 20%diH₂O (designated herein as W₂₀5GC5); 1% Cetylpyridinium Chloride, 5%TWEEN 20, 10% Glycerol, 64% Soybean Oil, and 20% diH₂O (designatedherein as W₂₀5GC); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol,64% Olive Oil, and 22% diH₂O (designated herein as W₂₀5EC Olive Oil); 1%Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64% Flaxseed Oil, and22% diH₂O (designated herein as W₂₀5EC Flaxseed Oil); 1% CetylpyridiniumChloride, 5% TWEEN 20, 8% Ethanol, 64% Corn Oil, and 22% diH₂O(designated herein as W₂₀5EC Corn Oil); 1% Cetylpyridinium Chloride, 5%TWEEN 20, 8% Ethanol, 64% Coconut Oil, and 22% diH₂O (designated hereinas W₂₀5EC Coconut Oil); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8%Ethanol, 64% Cottonseed Oil, and 22% diH₂O (designated herein as W₂₀5ECCottonseed Oil); 8% Dextrose, 5% TWEEN 10, 1% Cetylpyridinium Chloride,64% Soybean Oil, and 22% diH₂O (designated herein as W₂₀5C Dextrose); 8%PEG 200, 5% TWEEN 10, 1% Cetylpyridinium Chloride, 64% Soybean Oil, and22% diH₂O (designated herein as W₂₀5C PEG 200); 8% Methanol, 5% TWEEN10, 1% Cetylpyridinium Chloride, 64% Soybean Oil, and 22% diH₂O(designated herein as W₂₀5C Methanol); 8% PEG 1000, 5% TWEEN 10, 1%Cetylpyridinium Chloride, 64% Soybean Oil, and 22% diH₂O (designatedherein as W₂₀5C PEG 1000); 2% W₂₀5EC, 2% Natrosol 250H NF, and 96% diH₂O(designated herein as 2% W₂₀5EC Natrosol 2, also called 2% W₂₀5EC GEL);2% W₂₀5EC, 1% Natrosol 250H NF, and 97% diH₂O (designated herein as 2%W₂₀5EC Natrosol 1); 2% W₂₀5EC, 3% Natrosol 250H NF, and 95% diH₂O(designated herein as 2% W₂₀5EC Natrosol 3); 2% W₂₀5EC, 0.5% Natrosol250H NF, and 97.5% diH₂O (designated herein as 2% W₂₀5EC Natrosol 0.5);2% W₂₀5EC, 2% Methocel A, and 96% diH₂O (designated herein as 2% W₂₀5ECMethocel A); 2% W₂₀5EC, 2% Methocel K, and 96% diH₂O (designated hereinas 2% W₂₀5EC Methocel K); 2% Natrosol, 0.1% X8PC, 0.1×PBS, 5 mML-alanine, 5 mM Inosine, 10 mM Ammonium Chloride, and diH₂O (designatedherein as 0.1% X8PC/GE+2% Natrosol); 2% Natrosol, 0.8% Triton X-100,0.8% Tributyl Phosphate, 6.4% Soybean Oil, 0.1% CetylpyridiniumChloride, 0.1x PBS, 5 mM L-alanine, 5 mM Inosine, 10 mM AmmoniumChloride, and diH₂O (designated herein as 10% X8PC/GE+2% Natrosol); 1%Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64% Lard, and 22%diH₂O (designated herein as W₂₀5EC Lard); 1% Cetylpyridinium Chloride,5% TWEEN 20, 8% Ethanol, 64% Mineral Oil, and 22% diH₂O (designatedherein as W₂₀5EC Mineral Oil); 0.1% Cetylpyridinium Chloride, 2%Nerolidol, 5% TWEEN 20, 10% Ethanol, 64% Soybean Oil, and 18.9% diH₂O(designated herein as W₂₀5EC_(0.1)N); 0.1% Cetylpyridinium Chloride, 2%Farnesol, 5% TWEEN 20, 10% Ethanol, 64% Soybean Oil, and 18.9% diH₂O(designated herein as W₂₀SEC_(0.1)F); 0.1% Cetylpyridinium Chloride, 5%TWEEN 20, 10% Ethanol, 64% Soybean Oil, and 20.9% diH₂O (designatedherein as W₂₀5EC_(0.1)); 10% Cetylpyridinium Chloride, 8% TributylPhosphate, 8% Triton X-100, 54% Soybean Oil, and 20% diH₂O (designatedherein as X8PC₁₀); 5% Cetylpyridinium Chloride, 8% Triton X-100, 8%Tributyl Phosphate, 59% Soybean Oil, and 20% diH₂O (designated herein asX8PC₅); 0.02% Cetylpyridinium Chloride, 0.1% TWEEN 20, 10% Ethanol, 70%Soybean Oil, and 19.88% diH₂O (designated herein as W₂₀0.1EC_(0.02)); 1%Cetylpyridinium Chloride, 5% TWEEN 20, 8% Glycerol, 64% Mobil 1, and 22%diH₂O (designated herein as W₂₀5GC Mobil 1); 7.2% Triton X-100, 7.2%Tributyl Phosphate, 0.9% Cetylpyridinium Chloride, 57.6% Soybean Oil,0.1×PBS, 5 mM L-alanine, 5 mM Inosine, 10 mM Ammonium Chloride, and25.87% diH₂O (designated herein as 90% X8PC/GE); 7.2% Triton X-100, 7.2%Tributyl Phosphate, 0.9% Cetylpyridinium Chloride, 57.6% Soybean Oil, 1%EDTA, 5 mM L-alanine, 5 mM Inosine, 10 mM Ammonium Chloride, 0.1x PBS,and diH₂O (designated herein as 90% X8PC/GE EDTA); and 7.2% TritonX-100, 7.2% Tributyl Phosphate, 0.9% Cetylpyridinium Chloride, 57.6%Soybean Oil, 1% Sodium Thiosulfate, 5 mM L-alanine, 5 mM Inosine, 10 mMAmmonium Chloride, 0.1x PBS, and diH₂O (designated herein as 90% X8PC/GESTS).

In preferred embodiments of the present invention, the nanoemulsions arenon-toxic (e.g., to humans, plants, or animals), non-irritant (e.g., tohumans, plants, or animals), and non-corrosive (e.g., to humans, plants,or animals or the environment), while retaining stability when mixedwith other agents (e.g., a composition comprising an immunogen (e.g.,bacteria, fungi, viruses, and spores). While a number of the abovedescribed nanoemulsions meet these qualifications, the followingdescription provides a number of preferred non-toxic, non-irritant,non-corrosive, anti-microbial nanoemulsions of the present invention(hereinafter in this section referred to as “non-toxic nanoemulsions”).

In some embodiments the non-toxic nanoemulsions comprise surfactantlipid preparations (SLPs) for use as broad-spectrum antimicrobial agentsthat are effective against bacteria and their spores, enveloped viruses,and fungi. In preferred embodiments, these SLPs comprise a mixture ofoils, detergents, solvents, and cationic halogen-containing compounds inaddition to several ions that enhance their biocidal activities. TheseSLPs are characterized as stable, non-irritant, and non-toxic compoundscompared to commercially available bactericidal and sporicidal agents,which are highly irritant and/or toxic.

Ingredients for use in the non-toxic nanoemulsions include, but are notlimited to: detergents (e.g., TRITON X-100 (5-15%) or other members ofthe TRITON family, TWEEN 60 (0.5-2%) or other members of the TWEENfamily, or TYLOXAPOL (1-10%)); solvents (e.g., tributyl phosphate(5-15%)); alcohols (e.g., ethanol (5-15%) or glycerol (5-15%)); oils(e.g., soybean oil (40-70%)); cationic halogen-containing compounds(e.g., cetylpyridinium chloride (0.5-2%), cetylpyridinium bromide(0.5-2%)), or cetyldimethylethyl ammonium bromide (0.5-2%)); quaternaryammonium compounds (e.g., benzalkonium chloride (0.5-2%),N-alkyldimethylbenzyl ammonium chloride (0.5-2%)); ions (calciumchloride (1 mM-40 mM), ammonium chloride (1 mM-20 mM), sodium chloride(5 mM-200 mM), sodium phosphate (1 mM-20 mM)); nucleosides (e.g.,inosine (50 μM-20 mM)); and amino acids (e.g., L-alanine (50 μM-20 mM)).Emulsions are prepared, for example, by mixing in a high shear mixer for3-10 minutes. The emulsions may or may not be heated before mixing at82° C. for 1 hour.

Quaternary ammonium compounds for use in the present include, but arenot limited to, N-alkyldimethyl benzyl ammonium saccharinate;1,3,5-Triazine-1,3,5(2H, 4H, 6H)-triethanol; 1-Decanaminium,N-decyl-N,N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride;2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride; alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazoliniumchloride; alkyl bis(2-hydroxyethyl)benzyl ammonium chloride; alkyldemethyl benzyl ammonium chloride; alkyl dimethyl 3,4-dichlorobenzylammonium chloride (100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (50% C14, 40% C12, 10% C16); alkyl dimethyl 3,4-dichlorobenzylammonium chloride (55% C14, 23% C12, 20% C16); alkyl dimethyl benzylammonium chloride; alkyl dimethyl benzyl ammonium chloride (100% C14);alkyl dimethyl benzyl ammonium chloride (100% C16); alkyl dimethylbenzyl ammonium chloride (41% C14, 28% C12); alkyl dimethyl benzylammonium chloride (47% C12, 18% C14); alkyl dimethyl benzyl ammoniumchloride (55% C16, 20% C14); alkyl dimethyl benzyl ammonium chloride(58% C14, 28% C16); alkyl dimethyl benzyl ammonium chloride (60% C14,25% C12); alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14);alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyldimethyl benzyl ammonium chloride (65% C12, 25% C14); alkyl dimethylbenzyl ammonium chloride (67% C12, 24% C14); alkyl dimethyl benzylammonium chloride (67% C12, 25% C14); alkyl dimethyl benzyl ammoniumchloride (90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride (93%C14, 4% C12); alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18);alkyl dimethyl benzyl ammonium chloride (and) didecyl dimethyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids);alkyl dimethyl benzyl ammonium chloride (C12-C16); alkyl dimethyl benzylammonium chloride (C12-C18); alkyl dimethyl benzyl and dialkyl dimethylammonium chloride; alkyl dimethyl dimethybenzyl ammonium chloride; alkyldimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyldimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as inthe fatty acids of soybean oil); alkyl dimethyl ethylbenzyl ammoniumchloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14); alkyldimethyl isoproylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3%C18); alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1%C12); alkyl trimethyl ammonium chloride (90% C18, 10% C16);alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18); Di-(C8-10)-alkyldimethyl ammonium chlorides; dialkyl dimethyl ammonium chloride; dialkyldimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkylmethyl benzyl ammonium chloride; didecyl dimethyl ammonium chloride;diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammoniumchloride; dodecyl bis(2-hydroxyethyl) octyl hydrogen ammonium chloride;dodecyl dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyldimethyl benzyl ammonium chloride; heptadecyl hydroxyethylimidazoliniumchloride; hexahydro-1,3,5-thris(2-hydroxyethyl)-s-triazine;myristalkonium chloride (and) Quat RNIUM 14;N,N-Dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethylbenzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl ammoniumchloride; n-tetradecyl dimethyl benzyl ammonium chloride monohydrate;octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammoniumchloride; octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride;oxydiethylenebis (alkyl dimethyl ammonium chloride); quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride; trimethoxysily propyldimethyl octadecyl ammonium chloride; trimethoxysilyl quats, trimethyldodecylbenzyl ammonium chloride; n-dodecyl dimethyl ethylbenzyl ammoniumchloride; n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyldimethyl benzyl ammonium chloride; n-tetradecyl dimethyl ethyylbenzylammonium chloride; and n-octadecyl dimethyl benzyl ammonium chloride.

1. Aqueous Phase

In some embodiments, the emulsion comprises an aqueous phase. In certainpreferred embodiments, the emulsion comprises about 5 to 50, preferably10 to 40, more preferably 15 to 30, vol. % aqueous phase, based on thetotal volume of the emulsion (although other concentrations are alsocontemplated). In preferred embodiments, the aqueous phase compriseswater at a pH of about 4 to 10, preferably about 6 to 8. The water ispreferably deionized (hereinafter “DiH₂O”). In some embodiments, theaqueous phase comprises phosphate buffered saline (PBS). In somepreferred embodiments, the aqueous phase is sterile and pyrogen free.

2. Oil Phase

In some embodiments, the emulsion comprises an oil phase. In certainpreferred embodiments, the oil phase (e.g., carrier oil) of the emulsionof the present invention comprises 30-90, preferably 60-80, and morepreferably 60-70, vol. % of oil, based on the total volume of theemulsion (although higher and lower concentrations also find use inemulsions described herein).

The oil in the nanoemulsion adjuvant of the invention can be anycosmetically or pharmaceutically acceptable oil. The oil can be volatileor non-volatile, and may be chosen from animal oil, vegetable oil,natural oil, synthetic oil, hydrocarbon oils, silicone oils,semi-synthetic derivatives thereof, and combinations thereof.

Suitable oils include, but are not limited to, mineral oil, squaleneoil, flavor oils, silicon oil, essential oils, water insoluble vitamins,Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate,Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthylanthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate,neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyladipate, C₁₂₋₁₅ alkyl lactates, Cetyl lactate, Lauryl lactate,Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate,Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin, Fluidparaffins, Isododecane, Petrolatum, Argan oil, Canola oil, Chile oil,Coconut oil, corn oil, Cottonseed oil, Flaxseed oil, Grape seed oil,Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seedoil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Teaoil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil(simmondsia chinensis seed oil), Grapeseed oil, Macadamia oil, Wheatgerm oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil,Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nutoil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniperoil, seed oil, almond seed oil, anise seed oil, celery seed oil, cuminseed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil,cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemongrass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leafoil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmintleaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil,flower oil, chamomile oil, clary sage oil, clove oil, geranium floweroil, hyssop flower oil, jasmine flower oil, lavender flower oil, manukaflower oil, Marhoram flower oil, orange flower oil, rose flower oil,ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil,sassafras Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewoodoil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincenseoil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemonpeel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil,valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearylalcohol, semi-synthetic derivatives thereof, and any combinationsthereof.

The oil may further comprise a silicone component, such as a volatilesilicone component, which can be the sole oil in the silicone componentor can be combined with other silicone and non-silicone, volatile andnon-volatile oils. Suitable silicone components include, but are notlimited to, methylphenylpolysiloxane, simethicone, dimethicone,phenyltrimethicone (or an organomodified version thereof), alkylatedderivatives of polymeric silicones, cetyl dimethicone, lauryltrimethicone, hydroxylated derivatives of polymeric silicones, such asdimethiconol, volatile silicone oils, cyclic and linear silicones,cyclomethicone, derivatives of cyclomethicone,hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane,decamethylcyclopentasiloxane, volatile linear dimethylpolysiloxanes,isohexadecane, isoeicosane, isotetracosane, polyisobutene, isooctane,isododecane, semi-synthetic derivatives thereof, and combinationsthereof.

The volatile oil can be the organic solvent, or the volatile oil can bepresent in addition to an organic solvent. Suitable volatile oilsinclude, but are not limited to, a terpene, monoterpene, sesquiterpene,carminative, azulene, menthol, camphor, thujone, thymol, nerol,linalool, limonene, geraniol, perillyl alcohol, nerolidol, farnesol,ylangene, bisabolol, farnesene, ascaridole, chenopodium oil,citronellal, citral, citronellol, chamazulene, yarrow, guaiazulene,chamomile, semi-synthetic derivatives, or combinations thereof.

In one aspect of the invention, the volatile oil in the siliconecomponent is different than the oil in the oil phase.

In some embodiments, the oil phase comprises 3-15, and preferably 5-10vol. % of an organic solvent, based on the total volume of the emulsion.While the present invention is not limited to any particular mechanism,it is contemplated that the organic phosphate-based solvents employed inthe emulsions serve to remove or disrupt the lipids in the membranes ofthe pathogens. Thus, any solvent that removes the sterols orphospholipids in the microbial membranes finds use in the methods of thepresent invention. Suitable organic solvents include, but are notlimited to, organic phosphate based solvents or alcohols. In somepreferred embodiments, non-toxic alcohols (e.g., ethanol) are used as asolvent. The oil phase, and any additional compounds provided in the oilphase, are preferably sterile and pyrogen free.

3. Surfactants and Detergents

In some embodiments, the emulsions further comprises a surfactant ordetergent. In some preferred embodiments, the emulsion comprises fromabout 3 to 15%, and preferably about 10% of one or more surfactants ordetergents (although other concentrations are also contemplated). Whilethe present invention is not limited to any particular mechanism, it iscontemplated that surfactants, when present in the emulsions, help tostabilize the emulsions. Both non-ionic (non-anionic) and ionicsurfactants are contemplated. Additionally, surfactants from the BRIJfamily of surfactants find use in the compositions of the presentinvention. The surfactant can be provided in either the aqueous or theoil phase. Surfactants suitable for use with the emulsions include avariety of anionic and nonionic surfactants, as well as otheremulsifying compounds that are capable of promoting the formation ofoil-in-water emulsions. In general, emulsifying compounds are relativelyhydrophilic, and blends of emulsifying compounds can be used to achievethe necessary qualities. In some formulations, nonionic surfactants haveadvantages over ionic emulsifiers in that they are substantially morecompatible with a broad pH range and often form more stable emulsionsthan do ionic (e.g., soap-type) emulsifiers.

The surfactant in the nanoemulsion adjuvant of the invention can be apharmaceutically acceptable ionic surfactant, a pharmaceuticallyacceptable nonionic surfactant, a pharmaceutically acceptable cationicsurfactant, a pharmaceutically acceptable anionic surfactant, or apharmaceutically acceptable zwitterionic surfactant.

Exemplary useful surfactants are described in Applied Surfactants:Principles and Applications. Tharwat F. Tadros, Copyright 8 2005WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30629-3), whichis specifically incorporated by reference. Further, the surfactant canbe a pharmaceutically acceptable ionic polymeric surfactant, apharmaceutically acceptable nonionic polymeric surfactant, apharmaceutically acceptable cationic polymeric surfactant, apharmaceutically acceptable anionic polymeric surfactant, or apharmaceutically acceptable zwitterionic polymeric surfactant. Examplesof polymeric surfactants include, but are not limited to, a graftcopolymer of a poly(methyl methacrylate) backbone with multiple (atleast one) polyethylene oxide (PEO) side chain, polyhydroxystearic acid,an alkoxylated alkyl phenol formaldehyde condensate, a polyalkyleneglycol modified polyester with fatty acid hydrophobes, a polyester,semi-synthetic derivatives thereof, or combinations thereof.

Surface active agents or surfactants, are amphipathic molecules thatconsist of a non-polar hydrophobic portion, usually a straight orbranched hydrocarbon or fluorocarbon chain containing 8-18 carbon atoms,attached to a polar or ionic hydrophilic portion. The hydrophilicportion can be nonionic, ionic or zwitterionic. The hydrocarbon chaininteracts weakly with the water molecules in an aqueous environment,whereas the polar or ionic head group interacts strongly with watermolecules via dipole or ion-dipole interactions. Based on the nature ofthe hydrophilic group, surfactants are classified into anionic,cationic, zwitterionic, nonionic and polymeric surfactants.

Suitable surfactants include, but are not limited to, ethoxylatednonylphenol comprising 9 to 10 units of ethyleneglycol, ethoxylatedundecanol comprising 8 units of ethyleneglycol, polyoxyethylene (20)sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate,polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20)sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate,sorbitan monostearate, sorbitan monooleate, ethoxylated hydrogenatedricin oils, sodium laurylsulfate, a diblock copolymer of ethyleneoxydeand propyleneoxyde, Ethylene Oxide-Propylene Oxide Block Copolymers, andtetra-functional block copolymers based on ethylene oxide and propyleneoxide, Glyceryl monoesters, Glyceryl caprate, Glyceryl caprylate,Glyceryl cocate, Glyceryl erucate, Glyceryl hydroxysterate, Glycerylisostearate, Glyceryl lanolate, Glyceryl laurate, Glyceryl linolate,Glyceryl myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate,Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thighlycolate,Glyceryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate, Glyceryldisterate, Glyceryl sesuioleate, Glyceryl stearate lactate,Polyoxyethylene cetyl/stearyl ether, Polyoxyethylene cholesterol ether,Polyoxyethylene laurate or dilaurate, Polyoxyethylene stearate ordistearate, polyoxyethylene fatty ethers, Polyoxyethylene lauryl ether,Polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, asteroid, Cholesterol, Betasitosterol, Bisabolol, fatty acid esters ofalcohols, isopropyl myristate, Aliphati-isopropyl n-butyrate, Isopropyln-hexanoate, Isopropyl n-decanoate, Isoproppyl palmitate, Octyldodecylmyristate, alkoxylated alcohols, alkoxylated acids, alkoxylated amides,alkoxylated sugar derivatives, alkoxylated derivatives of natural oilsand waxes, polyoxyethylene polyoxypropylene block copolymers,nonoxynol-14, PEG-8 laurate, PEG-6 Cocoamide, PEG-20 methylglucosesesquistearate, PEG40 lanolin, PEG-40 castor oil, PEG-40 hydrogenatedcastor oil, polyoxyethylene fatty ethers, glyceryl diesters,polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, andpolyoxyethylene lauryl ether, glyceryl dilaurate, glyceryl dimystate,glyceryl distearate, semi-synthetic derivatives thereof, or mixturesthereof.

Additional suitable surfactants include, but are not limited to,non-ionic lipids, such as glyceryl laurate, glyceryl myristate, glyceryldilaurate, glyceryl dimyristate, semi-synthetic derivatives thereof, andmixtures thereof.

In additional embodiments, the surfactant is a polyoxyethylene fattyether having a polyoxyethylene head group ranging from about 2 to about100 groups, or an alkoxylated alcohol having the structure R₅—(OCH₂CH₂)_(y) —OH, wherein R₅ is a branched or unbranched alkyl group havingfrom about 6 to about 22 carbon atoms and y is between about 4 and about100, and preferably, between about 10 and about 100. Preferably, thealkoxylated alcohol is the species wherein R₅ is a lauryl group and yhas an average value of 23.

In a different embodiment, the surfactant is an alkoxylated alcoholwhich is an ethoxylated derivative of lanolin alcohol. Preferably, theethoxylated derivative of lanolin alcohol is laneth-10, which is thepolyethylene glycol ether of lanolin alcohol with an averageethoxylation value of 10.

Nonionic surfactants include, but are not limited to, an ethoxylatedsurfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a fattyacid ethoxylated, a monoalkaolamide ethoxylated, a sorbitan esterethoxylated, a fatty amino ethoxylated, an ethylene oxide-propyleneoxide copolymer, Bis(polyethylene glycol bis[imidazoyl carbonyl]),nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij® 35,Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor®EL, Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine,n-Decyl alpha-D-glucopyranoside, Decyl beta-D-maltopyranoside,n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecylbeta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycolmonodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethyleneglycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethyleneglycol monododecyl ether, Hexaethylene glycol monohexadecyl ether,Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether, Igepal CA-630, Igepal CA-630,Methyl-6-O—(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethyleneglycol monododecyl ether, N-Nonanoyl-N-methylglucamine,N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether,Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecylether, Octaethylene glycol monooctadecyl ether, Octaethylene glycolmonotetradecyl ether, Octyl-beta-D-glucopyranoside, Pentaethylene glycolmonodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethyleneglycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctylether, Polyethylene glycol diglycidyl ether, Polyethylene glycol etherW-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether,Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate,Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl),Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillajabark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85,Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5,Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10,Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7,Tergitol, Type NP-9, Tergitol, Tergitol, Type TMN-10, Tergitol, TypeTMN-6, Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecylether, Tetraethylene glycol monododecyl ether, Tetraethylene glycolmonotetradecyl ether, Triethylene glycol monodecyl ether, Triethyleneglycol monododecyl ether, Triethylene glycol monohexadecyl ether,Triethylene glycol monooctyl ether, Triethylene glycol monotetradecylether, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, TritonGR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, TritonX-15, Triton X-151, Triton X-200, Triton X-207, Triton® X-100, Triton®X-114, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45,Triton® X-705-70, TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61,TWEEN® 65, TWEEN® 80, TWEEN® 81, TWEEN® 85, Tyloxapol, n-Undecylbeta-D-glucopyranoside, semi-synthetic derivatives thereof, orcombinations thereof.

In addition, the nonionic surfactant can be a poloxamer. Poloxamers arepolymers made of a block of polyoxyethylene, followed by a block ofpolyoxypropylene, followed by a block of polyoxyethylene. The averagenumber of units of polyoxyethylene and polyoxypropylene varies based onthe number associated with the polymer. For example, the smallestpolymer, Poloxamer 101, consists of a block with an average of 2 unitsof polyoxyethylene, a block with an average of 16 units ofpolyoxypropylene, followed by a block with an average of 2 units ofpolyoxyethylene. Poloxamers range from colorless liquids and pastes towhite solids. In cosmetics and personal care products, Poloxamers areused in the formulation of skin cleansers, bath products, shampoos, hairconditioners, mouthwashes, eye makeup remover and other skin and hairproducts. Examples of Poloxamers include, but are not limited to,Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122, Poloxamer123, Poloxamer 124, Poloxamer 181, Poloxamer 182, Poloxamer 183,Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235,Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335,Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer407, Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.

Suitable cationic surfactants include, but are not limited to, aquarternary ammonium compound, an alkyl trimethyl ammonium chloridecompound, a dialkyl dimethyl ammonium chloride compound, a cationichalogen-containing compound, such as cetylpyridinium chloride,Benzalkonium chloride, Benzalkonium chloride,Benzyldimethylhexadecylammonium chloride,Benzyldimethyltetradecylammonium chloride, Benzyldodecyldimethylammoniumbromide, Benzyltrimethylammonium tetrachloroiodate,Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammoniumbromide, Dodecyltrimethylammonium bromide, Dodecyltrimethylammoniumbromide, Ethylhexadecyldimethylammonium bromide, Girard's reagent T,Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium bromide,N,N′,N′-Polyoxyethylene(10)-N-tallow-1,3-diaminopropane, Thonzoniumbromide, Trimethyl(tetradecyl)ammonium bromide, 1,3,5-Triazine-1,3,5(2H,4H, 6H)-triethanol, 1-Decanaminium, N-decyl-N,N-dimethyl-, chloride,Didecyl dimethyl ammonium chloride,2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride, Alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazoliniumchloride, Alkyl bis(2-hydroxyethyl)benzyl ammonium chloride, Alkyldemethyl benzyl ammonium chloride, Alkyl dimethyl 3,4-dichlorobenzylammonium chloride (100% C12), Alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (50% C14, 40% C12, 10% C16), Alkyl dimethyl 3,4-dichlorobenzylammonium chloride (55% C14, 23% C12, 20% C16), Alkyl dimethyl benzylammonium chloride, Alkyl dimethyl benzyl ammonium chloride (100% C14),Alkyl dimethyl benzyl ammonium chloride (100% C16), Alkyl dimethylbenzyl ammonium chloride (41% C14, 28% C12), Alkyl dimethyl benzylammonium chloride (47% C12, 18% C14), Alkyl dimethyl benzyl ammoniumchloride (55% C16, 20% C14), Alkyl dimethyl benzyl ammonium chloride(58% C14, 28% C16), Alkyl dimethyl benzyl ammonium chloride (60% C14,25% C12), Alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14),Alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14), Alkyldimethyl benzyl ammonium chloride (65% C12, 25% C14), Alkyl dimethylbenzyl ammonium chloride (67% C12, 24% C14), Alkyl dimethyl benzylammonium chloride (67% C12, 25% C14), Alkyl dimethyl benzyl ammoniumchloride (90% C14, 5% C12), Alkyl dimethyl benzyl ammonium chloride (93%C14, 4% C12), Alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18),Alkyl dimethyl benzyl ammonium chloride, Alkyl didecyl dimethyl ammoniumchloride, Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzylammonium chloride (C12-16), Alkyl dimethyl benzyl ammonium chloride(C12-18), Alkyl dimethyl benzyl ammonium chloride, dialkyl dimethylbenzyl ammonium chloride, Alkyl dimethyl dimethybenzyl ammoniumchloride, Alkyl dimethyl ethyl ammonium bromide (90% C14, 5% C16, 5%C12), Alkyl dimethyl ethyl ammonium bromide (mixed alkyl and alkenylgroups as in the fatty acids of soybean oil), Alkyl dimethyl ethylbenzylammonium chloride, Alkyl dimethyl ethylbenzyl ammonium chloride (60%C14), Alkyl dimethyl isopropylbenzyl ammonium chloride (50% C12, 30%C14, 17% C16, 3% C18), Alkyl trimethyl ammonium chloride (58% C18, 40%C16, 1% C14, 1% C12), Alkyl trimethyl ammonium chloride (90% C18, 10%C16), Alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18),Di-(C8-10)-alkyl dimethyl ammonium chlorides, Dialkyl dimethyl ammoniumchloride, Dialkyl methyl benzyl ammonium chloride, Didecyl dimethylammonium chloride, Diisodecyl dimethyl ammonium chloride, Dioctyldimethyl ammonium chloride, Dodecyl bis(2-hydroxyethyl) octyl hydrogenammonium chloride, Dodecyl dimethyl benzyl ammonium chloride,Dodecylcarbamoyl methyl dimethyl benzyl ammonium chloride, Heptadecylhydroxyethylimidazolinium chloride,Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine,Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Myristalkonium chloride(and) Quat RNIUM 14, N,N-Dimethyl-2-hydroxypropylammonium chloridepolymer, n-Tetradecyl dimethyl benzyl ammonium chloride monohydrate,Octyl decyl dimethyl ammonium chloride, Octyl dodecyl dimethyl ammoniumchloride, Octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride,Oxydiethylenebis(alkyl dimethyl ammonium chloride), Quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride, Trimethoxysily propyldimethyl octadecyl ammonium chloride, Trimethoxysilyl quats, Trimethyldodecylbenzyl ammonium chloride, semi-synthetic derivatives thereof, andcombinations thereof.

Exemplary cationic halogen-containing compounds include, but are notlimited to, cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides, ortetradecyltrimethylammonium halides. In some particular embodiments,suitable cationic halogen containing compounds comprise, but are notlimited to, cetylpyridinium chloride (CPC), cetyltrimethylammoniumchloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide(CPB), cetyltrimethylammonium bromide (CTAB), cetyidimethylethylammoniumbromide, cetyltributylphosphonium bromide, dodecyltrimethylammoniumbromide, and tetrad ecyltrimethylammonium bromide. In particularlypreferred embodiments, the cationic halogen containing compound is CPC,although the compositions of the present invention are not limited toformulation with an particular cationic containing compound.

Suitable anionic surfactants include, but are not limited to, acarboxylate, a sulphate, a sulphonate, a phosphate, chenodeoxycholicacid, chenodeoxycholic acid sodium salt, cholic acid, ox or sheep bile,Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid, Deoxycholic acidmethyl ester, Digitonin, Digitoxigenin, N,N-DimethyldodecylamineN-oxide, Docusate sodium salt, Glycochenodeoxycholic acid sodium salt,Glycocholic acid hydrate, synthetic, Glycocholic acid sodium salthydrate, synthetic, Glycodeoxycholic acid monohydrate, Glycodeoxycholicacid sodium salt, Glycodeoxycholic acid sodium salt, Glycolithocholicacid 3-sulfate disodium salt, Glycolithocholic acid ethyl ester,N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine solution,N-Lauroylsarcosine solution, Lithium dodecyl sulfate, Lithium dodecylsulfate, Lithium dodecyl sulfate, Lugol solution, Niaproof 4, Type 4,1-Octanesulfonic acid sodium salt, Sodium 1-butanesulfonate, Sodium1-decanesulfonate, Sodium 1-decanesulfonate, Sodium 1-dodecanesulfonate,Sodium 1-heptanesulfonate anhydrous, Sodium 1-heptanesulfonateanhydrous, Sodium 1-nonanesulfonate, Sodium 1-propanesulfonatemonohydrate, Sodium 2-bromoethanesulfonate, Sodium cholate hydrate,Sodium choleate, Sodium deoxycholate, Sodium deoxycholate monohydrate,Sodium dodecyl sulfate, Sodium hexanesulfonate anhydrous, Sodium octylsulfate, Sodium pentanesulfonate anhydrous, Sodium taurocholate,Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid sodiumsalt monohydrate, Taurohyodeoxycholic acid sodium salt hydrate,Taurolithocholic acid 3-sulfate disodium salt, Tauroursodeoxycholic acidsodium salt, Trizma® dodecyl sulfate, TWEEN® 80, Ursodeoxycholic acid,semi-synthetic derivatives thereof, and combinations thereof.

Suitable zwitterionic surfactants include, but are not limited to, anN-alkyl betaine, lauryl amindo propyl dimethyl betaine, an alkyldimethyl glycinate, an N-alkyl amino propionate, CHAPS, minimum 98%(TLC), CHAPS, SigmaUltra, minimum 98% (TLC), CHAPS, for electrophoresis,minimum 98% (TLC), CHAPSO, minimum 98%, CHAPSO, SigmaUltra, CHAPSO, forelectrophoresis, 3-(Decyldimethylammonio)propanesulfonate inner salt,3-Dodecyldimethylammonio)propanesulfonate inner salt, SigmaUltra,3-(Dodecyldimethylammonio)propanesulfonate inner salt,3-(N,N-Dimethylmyristylammonio)propanesulfonate,3-(N,N-Dimethyloctadecylammonio)propanesulfonate,3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,3-(N,N-Dimethylpalmitylammonio)propanesulfonate, semi-syntheticderivatives thereof, and combinations thereof.

The present invention is not limited to the surfactants disclosedherein. Additional surfactants and detergents useful in the compositionsof the present invention may be ascertained from reference works (e.g.,including, but not limited to, McCutheon's Volume 1: Emulsions andDetergents—North American Edition, 2000) and commercial sources.

4. Cationic Halogens Containg Compounds

In some embodiments, the emulsions further comprise a cationic halogencontaining compound. In some preferred embodiments, the emulsioncomprises from about 0.5 to 1.0 wt. % or more of a cationic halogencontaining compound, based on the total weight of the emulsion (althoughother concentrations are also contemplated). In preferred embodiments,the cationic halogen-containing compound is preferably premixed with theoil phase; however, it should be understood that the cationichalogen-containing compound may be provided in combination with theemulsion composition in a distinct formulation. Suitable halogencontaining compounds may be selected from compounds comprising chloride,fluoride, bromide and iodide ions. In preferred embodiments, suitablecationic halogen containing compounds include, but are not limited to,cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides, ortetradecyltrimethylammonium halides. In some particular embodiments,suitable cationic halogen containing compounds comprise, but are notlimited to, cetylpyridinium chloride (CPC), cetyltrimethylammoniumchloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide(CPB), and cetyltrimethylammonium bromide (CTAB),cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide,dodecyltrimethylammonium bromide, and tetrad ecyltrimethylammoniumbromide. In particularly preferred embodiments, the cationichalogen-containing compound is CPC, although the compositions of thepresent invention are not limited to formulation with any particularcationic containing compound.

5. Germination Enhancers

In other embodiments of the present invention, the nanoemulsions furthercomprise a germination enhancer. In some preferred embodiments, theemulsions comprise from about 1 mM to 15 mM, and more preferably fromabout 5 mM to 10 mM of one or more germination enhancing compounds(although other concentrations are also contemplated). In preferredembodiments, the germination enhancing compound is provided in theaqueous phase prior to formation of the emulsion. The present inventioncontemplates that when germination enhancers are added to thenanoemulsion compositions, the sporicidal properties of thenanoemulsions are enhanced. The present invention further contemplatesthat such germination enhancers initiate sporicidal activity nearneutral pH (between pH 6-8, and preferably 7). Such neutral pH emulsionscan be obtained, for example, by diluting with phosphate buffer saline(PBS) or by preparations of neutral emulsions. The sporicidal activityof the nanoemulsion preferentially occurs when the spores initiategermination.

In specific embodiments, it has been demonstrated that the emulsionsutilized in the vaccines of the present invention have sporicidalactivity. While the present invention is not limited to any particularmechanism and an understanding of the mechanism is not required topractice the present invention, it is believed that the fusigeniccomponent of the emulsions acts to initiate germination and beforereversion to the vegetative form is complete the lysogenic component ofthe emulsion acts to lyse the newly germinating spore. These componentsof the emulsion thus act in concert to leave the spore susceptible todisruption by the emulsions. The addition of germination enhancerfurther facilitates the anti-sporicidal activity of the emulsions, forexample, by speeding up the rate at which the sporicidal activityoccurs.

Germination of bacterial endospores and fungal spores is associated withincreased metabolism and decreased resistance to heat and chemicalreactants. For germination to occur, the spore must sense that theenvironment is adequate to support vegetation and reproduction. Theamino acid L-alanine stimulates bacterial spore germination (See e.g.,Hills, J. Gen. Micro. 4:38 (1950); and Halvorson and Church, BacteriolRev. 21:112 (1957)). L-alanine and L-proline have also been reported toinitiate fungal spore germination (Yanagita, Arch Mikrobiol 26:329(1957)). Simple α-amino acids, such as glycine and L-alanine, occupy acentral position in metabolism. Transamination or deamination of α-aminoacids yields the glycogenic or ketogenic carbohydrates and the nitrogenneeded for metabolism and growth. For example, transamination ordeamination of L-alanine yields pyruvate, which is the end product ofglycolytic metabolism (EmBECTON DICKENSONen-Meyerhof-Parnas Pathway).Oxidation of pyruvate by pyruvate dehydrogenase complex yieldsacetyl-CoA, NADH, H⁺, and CO₂. Acetyl-CoA is the initiator substrate forthe tricarboxylic acid cycle (Kreb's Cycle), which in turns feeds themitochondrial electron transport chain. Acetyl-CoA is also the ultimatecarbon source for fatty acid synthesis as well as for sterol synthesis.Simple α-amino acids can provide the nitrogen, CO₂, glycogenic and/orketogenic equivalents required for germination and the metabolicactivity that follows.

In certain embodiments, suitable germination enhancing agents of theinvention include, but are not limited to, -amino acids comprisingglycine and the L-enantiomers of alanine, valine, leucine, isoleucine,serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl estersthereof. Additional information on the effects of amino acids ongermination may be found in U.S. Pat. No. 5,510,104; herein incorporatedby reference in its entirety. In some embodiments, a mixture of glucose,fructose, asparagine, sodium chloride (NaCl), ammonium chloride (NH₄Cl),calcium chloride (CaCl₂) and potassium chloride (KCl) also may be used.In particularly preferred embodiments of the present invention, theformulation comprises the germination enhancers L-alanine, CaCl₂,Inosine and NH₄Cl. In some embodiments, the compositions furthercomprise one or more common forms of growth media (e.g., trypticase soybroth, and the like) that additionally may or may not itself comprisegermination enhancers and buffers.

The above compounds are merely exemplary germination enhancers and it isunderstood that other known germination enhancers will find use in thenanoemulsions utilized in some embodiments of the present invention. Acandidate germination enhancer should meet two criteria for inclusion inthe compositions of the present invention: it should be capable of beingassociated with the emulsions disclosed herein and it should increasethe rate of germination of a target spore when incorporated in theemulsions disclosed herein. One skilled in the art can determine whethera particular agent has the desired function of acting as an germinationenhancer by applying such an agent in combination with the nanoemulsionsdisclosed herein to a target and comparing the inactivation of thetarget when contacted by the admixture with inactivation of like targetsby the composition of the present invention without the agent. Any agentthat increases germination, and thereby decreases or inhibits the growthof the organisms, is considered a suitable enhancer for use in thenanoemulsion compositions disclosed herein.

In still other embodiments, addition of a germination enhancer (orgrowth medium) to a neutral emulsion composition produces a compositionthat is useful in inactivating bacterial spores in addition to envelopedviruses, Gram negative bacteria, and Gram positive bacteria for use inthe vaccine compositions of the present invention.

6. Interaction Enhancers

In still other embodiments, nanoemulsions comprise one or more compoundscapable of increasing the interaction of the compositions (i.e.,“interaction enhancer” (e.g., with target pathogens (e.g., the cell wallof Gram negative bacteria such as Vibrio, Salmonella, Shigella andPseudomonas)). In preferred embodiments, the interaction enhancer ispreferably premixed with the oil phase; however, in other embodimentsthe interaction enhancer is provided in combination with thecompositions after emulsification. In certain preferred embodiments, theinteraction enhancer is a chelating agent (e.g.,ethylenediaminetetraacetic acid (EDTA) orethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) in a buffer(e.g., tris buffer)). It is understood that chelating agents are merelyexemplary interaction enhancing compounds. Indeed, other agents thatincrease the interaction of the nanoemulsions used in some embodimentsof the present invention (e.g., with microbial agents, pathogens,vaccines, etc.) are contemplated. In particularly preferred embodiments,the interaction enhancer is at a concentration of about 50 to about 250μM. One skilled in the art will be able to determine whether aparticular agent has the desired function of acting as an interactionenhancer by applying such an agent in combination with the compositionsof the present invention to a target and comparing the inactivation ofthe target when contacted by the admixture with inactivation of liketargets by the composition of the present invention without the agent.Any agent that increases the interaction of an emulsion with bacteriaand thereby decreases or inhibits the growth of the bacteria, incomparison to that parameter in its absence, is considered aninteraction enhancer.

In some embodiments, the addition of an interaction enhancer tonanoemulsion produces a composition that is useful in inactivatingenveloped viruses, some Gram positive bacteria and some Gram negativebacteria for use in a vaccine composition.

7. Quaternary Ammonium Compounds

In some embodiments, nanoemulsions of the present invention include aquaternary ammonium containing compound. Exemplary quaternary ammoniumcompounds include, but are not limited to, Alkyl dimethyl benzylammonium chloride, didecyl dimethyl ammonium chloride, Alkyl dimethylbenzyl and dialkyl dimethyl ammonium chloride,N,N-Dimethyl-2-hydroxypropylammonium chloride polymer, Didecyl dimethylammonium chloride, n-Alkyl dimethyl benzyl ammonium chloride, n-Alkyldimethyl ethylbenzyl ammonium chloride,

Dialkyl dimethyl ammonium chloride, n-Alkyl dimethyl benzyl ammoniumchloride, n-Tetradecyl dimethyl benzyl ammonium chloride monohydrate,n-Alkyl dimethyl benzyl ammonium chloride, Dialkyl dimethyl ammoniumchloride, Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine,Myristalkonium chloride (and) Quat RNIUM 14, Alkylbis(2-hydroxyethyl)benzyl ammonium chloride, Alkyl demethyl benzylammonium chloride, Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride,Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzyldimethylbenzyl ammonium, Alkyl dimethyl dimethybenzyl ammonium chloride,Alkyl dimethyl ethyl ammonium bromide, Alkyl dimethyl ethyl ammoniumbromide, Alkyl dimethyl ethylbenzyl ammonium chloride, Alkyl dimethylisopropylbenzyl ammonium chloride, Alkyl trimethyl ammonium chloride,Alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazolinium chloride, Dialkylmethyl benzyl ammonium chloride, Dialkyl dimethyl ammonium chloride,Didecyl dimethyl ammonium chloride,2-(2-(p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride, 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride, Dioctyl dimethyl ammonium chloride, Dodecylbis(2-hydroxyethyl) octyl hydrogen ammonium chloride, Dodecyl dimethylbenzyl ammonium chloride, Dodecylcarbamoyl methyl dimethyl benzylammonium chloride, Heptadecyl hydroxyethylimidazolinium chloride,Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, Octyl decyl dimethylammonium chloride, Octyl dodecyl dimethyl ammonium chloride,Octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride,Oxydiethylenebis(alkyl dimethyl ammonium chloride), Quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride, Trimethoxysilyl quats, andTrimethyl dodecylbenzyl ammonium chloride.

8. Other Components

In some embodiments, a nanoemulsion adjuvant composition comprises oneor more additional components that provide a desired property orfunctionality to the nanoemulsions. These components may be incorporatedinto the aqueous phase or the oil phase of the nanoemulsions and/or maybe added prior to or following emulsification. For example, in someembodiments, the nanoemulsions further comprise phenols (e.g.,triclosan, phenyl phenol), acidifying agents (e.g., citric acid (e.g.,1.5-6%), acetic acid, lemon juice), alkylating agents (e.g., sodiumhydroxide (e.g., 0.3%)), buffers (e.g., citrate buffer, acetate buffer,and other buffers useful to maintain a specific pH), and halogens (e.g.,polyvinylpyrrolidone, sodium hypochlorite, hydrogen peroxide).

Exemplary techniques for making a nanoemulsion are described below.Additionally, a number of specific, although exemplary, formulationrecipes are also set forth herein.

In some embodiments, a nanoemulsion adjuvant is administered to asubject before, concurrent with or after administration of a compositioncomprising an immunogen (e.g., a pathogen and/or pathogen component(e.g., purified, isolated and/or recombinant pathogen peptide and/orprotein)). The invention is not limited to the use of any one specifictype of composition comprising an immunogen. Indeed, a variety ofcompositions comprising an immunogen (e.g., utilized for generating animmune response (e.g., for use as a vaccine)) may be utilized with ananoemulsion adjuvant of the invention. In some embodiments, thecomposition comprising an immunogen comprises pathogens (e.g., killedpathogens), pathogen components or isolated, purified and/or recombinantparts thereof.

In some embodiments, a nanoemulsion adjuvant is administered to asubject before, concurrent with or after administration of a vaccinecontaining peptides (e.g., one generally well known in the art, asexemplified by U.S. Pat. Nos. 4,601,903; 4,599,231; 4,599,230; and4,596,792; each of which is hereby incorporated by reference).

Formulation Techniques

Nanoemulsions of the present invention can be formed using classicemulsion forming techniques. In brief, the oil phase is mixed with theaqueous phase under relatively high shear forces (e.g., using highhydraulic and mechanical forces) to obtain an oil-in-water nanoemulsion.The emulsion is formed by blending the oil phase with an aqueous phaseon a volume-to-volume basis ranging from about 1:9 to 5:1, preferablyabout 5:1 to 3:1, most preferably 4:1, oil phase to aqueous phase. Theoil and aqueous phases can be blended using any apparatus capable ofproducing shear forces sufficient to form an emulsion such as FrenchPresses or high shear mixers (e.g., FDA approved high shear mixers areavailable, for example, from Admix, Inc., Manchester, N.H.). Methods ofproducing such emulsions are described in U.S. Pat. Nos. 5,103,497 and4,895,452, and U.S. Patent Application Nos. 20070036831, 20060251684,and 20050208083, herein incorporated by reference in their entireties.

In preferred embodiments, compositions used in the methods of thepresent invention comprise droplets of an oily discontinuous phasedispersed in an aqueous continuous phase, such as water. In preferredembodiments, nanoemulsions of the present invention are stable, and donot decompose even after long storage periods (e.g., greater than one ormore years). Furthermore, in some embodiments, nanoemulsions are stable(e.g., in some embodiments for greater than 3 months, in someembodiments for greater than 6 months, in some embodiments for greaterthan 12 months, in some embodiments for greater than 18 months) aftercombination with an immunogen. In preferred embodiments, nanoemulsionsof the present invention are non-toxic and safe when administered (e.g.,via spraying or contacting mucosal surfaces, swallowed, inhaled, etc.)to a subject.

In some embodiments, a portion of the emulsion may be in the form oflipid structures including, but not limited to, unilamellar,multilamellar, and paucliamellar lipid vesicles, micelles, and lamellarphases.

As described above, the present invention is not limited by the type ofsubject administered a composition of the present invention. Indeed, awide variety of subjects are contemplated to be benefited fromadministration of a composition of the present invention. In preferredembodiments, the subject is a human. In some embodiments, human subjectsare of any age (e.g., adults, children, infants, etc.) that have been orare likely to become exposed to a microorganism. In some embodiments,the human subjects are subjects that are more likely to receive a directexposure to pathogenic microorganisms or that are more likely to displaysigns and symptoms of disease after exposure to a pathogen (e.g.,subjects with CF or asthma, subjects in the armed forces, governmentemployees, frequent travelers, persons attending or working in a schoolor daycare, health care workers, an elderly person, an immunocompromisedperson, and emergency service employees (e.g., police, fire, EMTemployees)). In some embodiments, any one or all members of the generalpublic can be administered a composition of the present invention (e.g.,to prevent the occurrence or spread of disease). For example, in someembodiments, compositions and methods of the present invention areutilized to treat a group of people (e.g., a population of a region,city, state and/or country) for their own health (e.g., to prevent ortreat disease) and/or to prevent or reduce the risk of disease spreadfrom animals (e.g., birds, cattle, sheep, pigs, etc.) to humans. In someembodiments, the subjects are non-human mammals (e.g., pigs, cattle,goats, horses, sheep, or other livestock; or mice, rats, rabbits orother animal). In some embodiments, compositions and methods of thepresent invention are utilized in research settings (e.g., with researchanimals).

A composition comprising a nanoemulsion of the present invention can beadministered (e.g., to a subject (e.g., via pulmonary and/or mucosalroute)) as a therapeutic or as a prophylactic to prevent microbialinfection.

Therapeutics and Prophylactics

Furthermore, in preferred embodiments, a composition of the presentinvention induces (e.g., when administered to a subject) both systemicand mucosal immunity. Thus, in some preferred embodiments,administration of a composition comprising a nanoemulsion andStreptococcus (e.g., Streptococcus pneumoniae) antigen to a subjectresults in protection against an exposure (e.g., a mucosal exposure) toStreptococcus. Although an understanding of the mechanism is notnecessary to practice the present invention and the present invention isnot limited to any particular mechanism of action, mucosaladministration (e.g., vaccination) provides protection againstStreptococcus (e.g., Streptococcus pneumoniae) infection (e.g., thatinitiates at a mucosal surface). Although it has heretofore provendifficult to stimulate secretory IgA responses and protection againstpathogens that invade at mucosal surfaces (See, e.g., Mestecky et al,Mucosal Immunology. 3ed edn. (Academic Press, San Diego, 2005)), in someembodiments, the present invention provides compositions and methods forstimulating mucosal immunity (e.g., a protective IgA response) from apathogen (e.g., pathogenic species of Streptococcus (e.g., Streptococcuspneumoniae)) in a subject.

In some embodiments, the present invention provides a composition (e.g.,a composition comprising a nanoemulsion and Streptococcus (e.g.,Streptococcus pneumoniae) antigen) to serve as a mucosal vaccine. Insome embodiments, this material is produced with NE and killed wholecell bacteria of the genus Streptococcus (e.g., Streptococcus pneumoniae(e.g., killed using nanoemulsion, alcohol (e.g., ethanol), or othermethods), isolated, purified and/or recombinant protein and/orsaccharide component of Streptococcus (e.g., protein/peptide (e.g.,Streptococcus-derived protein, live-virus-vector-derived protein,recombinant protein, recombinant denatured protein/antigens, smallpeptide segments protein/antigen). The ability to produce thisformulation rapidly and administer it via mucosal (e.g., nasal)instillation provides a vaccine that can be used in large-scaleadministrations (e.g., to a population of a town, village, city, stateor country). The present invention is not limited to any particularformulation (e.g., comprising a nanoemulsion and one or more recombinantStreptococcus proteins). For example, in some embodiments, the inventionprovides a nanoemulsion described herein combined with one or morerecombinant Streptococcus proteins (e.g., PsaA, PiuA, PavA).

In some preferred embodiments, the present invention provides acomposition for generating an immune response comprising a NE and animmunogen (e.g., a purified, isolated or synthetic Streptococcus proteinor derivative, variant, or analogue thereof; or, one or more serotypesof Streptococcus (e.g., Streptococcus pneumoniae (e.g., killed and orinactivated whole cell bacteria). When administered to a subject, acomposition of the present invention stimulates an immune responseagainst the immunogen within the subject. Although an understanding ofthe mechanism is not necessary to practice the present invention and thepresent invention is not limited to any particular mechanism of action,in some embodiments, generation of an immune response (e.g., resultingfrom administration of a composition comprising a nanoemulsion and animmunogen) provides total or partial immunity to the subject (e.g., fromsigns, symptoms or conditions of a disease (e.g., strep throat,meningitis, bacterial pneumoniae, endocarditis, erysipelas and/ornecrotizing fasciitis)). Without being bound to any specific theory,protection and/or immunity from disease (e.g., the ability of asubject's immune system to prevent or attenuate (e.g., suppress) a sign,symptom or condition of disease) after exposure to an immunogeniccomposition of the present invention is due to adaptive (e.g., acquired)immune responses (e.g., immune responses mediated by B and T cellsfollowing exposure to a NE comprising an immunogen of the presentinvention (e.g., immune responses that exhibit increased specificity andreactivity towards Streptococcus (e.g., Streptococcus pneumoniae)).Thus, in some embodiments, the compositions and methods of the presentinvention are used prophylactically or therapeutically to prevent orattenuate a sign, symptom or condition associated with Streptococcus(e.g., Streptococcus pneumoniae)).

In some embodiments, a NE comprising an immunogen (e.g., a Streptococcus(e.g., Streptococcus pneumoniae) antigen) is administered alone. In someembodiments, a composition comprising a NE and an immunogen (e.g., aStreptococcus (e.g., Streptococcus pneumoniae) antigen) comprises one ormore other agents (e.g., a pharmaceutically acceptable carrier,adjuvant, excipient, and the like). In some embodiments, a compositionfor stimulating an immune response of the present invention isadministered in a manner to induce a humoral immune response. In someembodiments, a composition for stimulating an immune response of thepresent invention is administered in a manner to induce a cellular(e.g., cytotoxic T lymphocyte) immune response, rather than a humoralresponse. In some embodiments, a composition comprising a NE and animmunogen of the present invention induces both a cellular and humoralimmune response.

The present invention is not limited by the isotype or strain ofStreptococcus (e.g., Streptococcus pneumoniae) used in a compositioncomprising a NE and immunogen. Indeed, each Streptococcus (e.g.,Streptococcus pneumoniae) family member alone, or in combination withanother family member, may be used to generate a composition comprisinga NE and an immunogen (e.g., used to generate an immune response) of thepresent invention. Exemplary species of Streptococcus and isotypes ofStreptococcus pneumoniae are described herein.

Thus, in some embodiments, the Streptococcus (e.g., Streptococcuspneumoniae) strain utilized is a modified (e.g., genetically modified(e.g., naturally modified via natural selection or modified usingrecombinant genetic techniques)) strain that displays greater pathogeniccapacity (e.g., causes more sever Streptococcus—(e.g., Streptococcuspneumoniae)—induced disease (e.g., comprising enhanced and/or moresevere strep throat, meningitis, etc.)). In some embodiments, any one ormore members of the Streptococcus genus is utilized in an immunoreactivecomposition of the invention including but not limited to S. pneumoniae,S. mutans, S. mitis, S. sanguinis, S. salivarius, S. viridans, S.salivarius ssp. thermophilus, S. constellatus, S. pyogenes, S.agalactiae, S. zooepidemicus, Streptococcus bovis and Streptococcusequines, Streptococcus canis, as well as former Group D Streptococciincluding S. faecalis, S. faecium, S. durans, and S. avium.

The present invention is not limited by the Streptococcus (e.g.,Streptococcus pneumoniae) isotype and/or strain used. Indeed, a varietyof Streptococcus (e.g., Streptococcus pneumoniae) strains arecontemplated to be useful in the present invention including, but notlimited to, classical strains, attenuated strains, non-replicatingstrains, modified strains (e.g., genetically or mechanically modifiedstrains (e.g., to become more or less virulent)), or other seriallydiluted strains of Streptococcus (e.g., Streptococcus pneumoniae). Acomposition comprising a NE and immunogen may comprise one or morestrains of Streptococcus (e.g., Streptococcus pneumoniae) and/or othertype of Streptococcus (e.g., Streptococcus pneumoniae). Additionally, acomposition comprising a NE and immunogen may comprise one or morestrains of Streptococcus (e.g., Streptococcus pneumoniae), and, inaddition, one or more strains of a non-Streptococcus (e.g.,Streptococcus pneumoniae) immunogen.

In some embodiments, the immunogen may comprise one or more antigensderived from a pathogen (e.g., Streptococcus (e.g., Streptococcuspneumoniae)). For example, in some embodiments, the immunogen is apurified, recombinant, synthetic, or otherwise isolated protein (e.g.,added to the NE to generate an immunogenic composition). Similarly, theimmunogenic protein may be a derivative, analogue or otherwise modified(e.g., PEGylated) form of a protein from a pathogen.

The present invention is not limited by the particular formulation of acomposition comprising a NE and immunogen of the present invention.Indeed, a composition comprising a NE and immunogen of the presentinvention may comprise one or more different agents in addition to theNE and immunogen. These agents or cofactors include, but are not limitedto, adjuvants, surfactants, additives, buffers, solubilizers, chelators,oils, salts, therapeutic agents, drugs, bioactive agents,antibacterials, and antimicrobial agents (e.g., antibiotics, antivirals,etc.). In some embodiments, a composition comprising a NE and immunogenof the present invention comprises an agent and/or co-factor thatenhance the ability of the immunogen to induce an immune response (e.g.,an adjuvant). In some preferred embodiments, the presence of one or moreco-factors or agents reduces the amount of immunogen required forinduction of an immune response (e.g., a protective immune response(e.g., protective immunization)). In some embodiments, the presence ofone or more co-factors or agents can be used to skew the immune responsetowards a cellular (e.g., T cell mediated) or humoral (e.g., antibodymediated) immune response. The present invention is not limited by thetype of co-factor or agent used in a therapeutic agent of the presentinvention.

Adjuvants are described in general in Vaccine Design—the Subunit andAdjuvant Approach, edited by Powell and Newman, Plenum Press, New York,1995. The present invention is not limited by the type of adjuvantutilized (e.g., for use in a composition (e.g., pharmaceuticalcomposition) comprising a NE and immunogen). For example, in someembodiments, suitable adjuvants include an aluminum salt such asaluminum hydroxide gel (alum) or aluminum phosphate. In someembodiments, an adjuvant may be a salt of calcium, iron or zinc, or maybe an insoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatised polysaccharides, orpolyphosphazenes.

In some embodiments, it is preferred that a composition comprising a NEand immunogen of the present invention comprises one or more adjuvantsthat induce a Th1-type response. However, in other embodiments, it willbe preferred that a composition comprising a NE and immunogen of thepresent invention comprises one or more adjuvants that induce a Th2-typeresponse.

In general, an immune response is generated to an antigen through theinteraction of the antigen with the cells of the immune system. Immuneresponses may be broadly categorized into two categories: humoral andcell mediated immune responses (e.g., traditionally characterized byantibody and cellular effector mechanisms of protection, respectively).These categories of response have been termed Th1-type responses(cell-mediated response), and Th2-type immune responses (humoralresponse).

Stimulation of an immune response can result from a direct or indirectresponse of a cell or component of the immune system to an intervention(e.g., exposure to an immunogen). Immune responses can be measured inmany ways including activation, proliferation or differentiation ofcells of the immune system (e.g., B cells, T cells, dendritic cells,APCs, macrophages, NK cells, NKT cells etc.); up-regulated ordown-regulated expression of markers and cytokines; stimulation of IgA,IgM, or IgG titer; splenomegaly (including increased spleencellularity); hyperplasia and mixed cellular infiltrates in variousorgans. Other responses, cells, and components of the immune system thatcan be assessed with respect to immune stimulation are known in the art.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, compositions andmethods of the present invention induce expression and secretion ofcytokines (e.g., by macrophages, dendritic cells and CD4+ T cells).Modulation of expression of a particular cytokine can occur locally orsystemically. It is known that cytokine profiles can determine T cellregulatory and effector functions in immune responses. In someembodiments, Th1-type cytokines can be induced, and thus, theimmunostimulatory compositions of the present invention can promote aTh1 type antigen-specific immune response including cytotoxic T-cells(e.g., thereby avoiding unwanted Th2 type immune responses (e.g.,generation of Th2 type cytokines (e.g., IL-13) involved in enhancing theseverity of disease (e.g., IL-13 induction of mucus formation))).

Cytokines play a role in directing the T cell response. Helper (CD4+) Tcells orchestrate the immune response of mammals through production ofsoluble factors that act on other immune system cells, including B andother T cells. Most mature CD4+ T helper cells express one of twocytokine profiles: Th1 or Th2. Th1-type CD4+ T cells secrete IL-2, IL-3,IFN-γ, GM-CSF and high levels of TNF-α. Th2 cells express IL-3, IL-4,IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-α. Th1 typecytokines promote both cell-mediated immunity, and humoral immunity thatis characterized by immunoglobulin class switching to IgG2a in mice andIgG1 in humans. Th1 responses may also be associated with delayed-typehypersensitivity and autoimmune disease. Th2 type cytokines induceprimarily humoral immunity and induce class switching to IgG1 and IgE.The antibody isotypes associated with Th1 responses generally haveneutralizing and opsonizing capabilities whereas those associated withTh2 responses are associated more with allergic responses.

Several factors have been shown to influence skewing of an immuneresponse towards either a Th1 or Th2 type response. The bestcharacterized regulators are cytokines. IL-12 and IFN-γ are positive Th1and negative Th2 regulators. IL-12 promotes IFN-γ production, and IFN-γprovides positive feedback for IL-12. IL-4 and IL-10 appear importantfor the establishment of the Th2 cytokine profile and to down-regulateTh1 cytokine production.

Thus, in preferred embodiments, the present invention provides a methodof stimulating a Th1-type immune response in a subject comprisingadministering to a subject a composition comprising a NE and animmunogen. However, in other embodiments, the present invention providesa method of stimulating a Th2-type immune response in a subject (e.g.,if balancing of a T cell mediated response is desired) comprisingadministering to a subject a composition comprising a NE and animmunogen. In further preferred embodiments, adjuvants can be used(e.g., can be co-administered with a composition of the presentinvention) to skew an immune response toward either a Th1 or Th2 typeimmune response. For example, adjuvants that induce Th2 or weak Th1responses include, but are not limited to, alum, saponins, and SB-As4.Adjuvants that induce Th1 responses include but are not limited to MPL,MDP, ISCOMS, IL-12, IFN-γ, and SB-AS2.

Several other types of Th1-type immunogens can be used (e.g., as anadjuvant) in compositions and methods of the present invention. Theseinclude, but are not limited to, the following. In some embodiments,monophosphoryl lipid A (e.g., in particular 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL)), is used. 3D-MPL is a well knownadjuvant manufactured by Ribi Immunochem, Montana. Chemically it isoften supplied as a mixture of 3-de-O-acylated monophosphoryl lipid Awith either 4, 5, or 6 acylated chains. In some embodiments,diphosphoryl lipid A, and 3-O-deacylated variants thereof are used. Eachof these immunogens can be purified and prepared by methods described inGB 2122204B, hereby incorporated by reference in its entirety. Otherpurified and synthetic lipopolysaccharides have been described (See,e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et al., 1986,Int. Arch. Allergy. Immunol., 79(4):392-6; Hilgers et al., 1987,Immunology, 60(1):141-6; and EP 0 549 074, each of which is herebyincorporated by reference in its entirety). In some embodiments, 3D-MPLis used in the form of a particulate formulation (e.g., having a smallparticle size less than 0.2 μm in diameter, described in EP 0 689 454,hereby incorporated by reference in its entirety).

In some embodiments, saponins are used as an immunogen (e.g., Th1-typeadjuvant) in a composition of the present invention. Saponins are wellknown adjuvants (See, e.g., Lacaille-Dubois and Wagner (1996)Phytomedicine vol 2 pp 363-386). Examples of saponins include Quil A(derived from the bark of the South American tree Quillaja SaponariaMolina), and fractions thereof (See, e.g., U.S. Pat. No. 5,057,540;Kensil, Crit. Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0362 279, each of which is hereby incorporated by reference in itsentirety). Also contemplated to be useful in the present invention arethe haemolytic saponins QS7, QS17, and QS21 (HPLC purified fractions ofQuil A; See, e.g., Kensil et al. (1991). J. Immunology 146, 431-437,U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0 362 279, eachof which is hereby incorporated by reference in its entirety). Alsocontemplated to be useful are combinations of QS21 and polysorbate orcyclodextrin (See, e.g., WO 99/10008, hereby incorporated by referencein its entirety.

In some embodiments, an immunogenic oligonucleotide containingunmethylated CpG dinucleotides (“CpG”) is used as an adjuvant in thepresent invention. CpG is an abbreviation for cytosine-guanosinedinucleotide motifs present in DNA. CpG is known in the art as being anadjuvant when administered by both systemic and mucosal routes (See,e.g., WO 96/02555, EP 468520, Davis et al., J. Immunol, 1998,160(2):870-876; McCluskie and Davis, J. Immunol., 1998, 161(9):4463-6;and U.S. Pat. App. No. 20050238660, each of which is hereby incorporatedby reference in its entirety). For example, in some embodiments, theimmunostimulatory sequence is Purine-Purine-C-G-pyrimidine-pyrimidine;wherein the CG motif is not methylated.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, the presence of oneor more CpG oligonucleotides activate various immune subsets includingnatural killer cells (which produce IFN-γ) and macrophages. In someembodiments, CpG oligonucleotides are formulated into a composition ofthe present invention for inducing an immune response. In someembodiments, a free solution of CpG is co-administered together with anantigen (e.g., present within a NE solution (See, e.g., WO 96/02555;hereby incorporated by reference). In some embodiments, a CpGoligonucleotide is covalently conjugated to an antigen (See, e.g., WO98/16247, hereby incorporated by reference), or formulated with acarrier such as aluminium hydroxide (See, e.g., Brazolot-Millan et al.,Proc. Natl. Acad Sci., USA, 1998, 95(26), 15553-8).

In some embodiments, adjuvants such as Complete Freunds Adjuvant andIncomplete Freunds Adjuvant, cytokines (e.g., interleukins (e.g., IL-2,IFN-γ, IL-4, etc.), macrophage colony stimulating factor, tumor necrosisfactor, etc.), detoxified mutants of a bacterial ADP-ribosylating toxinsuch as a cholera toxin (CT), a pertussis toxin (PT), or an E. Coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (See, e.g.,WO93/13202 and WO92/19265, each of which is hereby incorporated byreference), and other immunogenic substances (e.g., that enhance theeffectiveness of a composition of the present invention) are used with acomposition comprising a NE and immunogen of the present invention.

Additional examples of adjuvants that find use in the present inventioninclude poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; VirusResearch Institute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); and Leishmania elongation factor (apurified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Adjuvants may be added to a composition comprising a NE and animmunogen, or, the adjuvant may be formulated with carriers, for exampleliposomes, or metallic salts (e.g., aluminium salts (e.g., aluminiumhydroxide)) prior to combining with or co-administration with acomposition comprising a NE and an immunogen.

In some embodiments, a composition comprising a NE and an immunogencomprises a single adjuvant. In other embodiments, a compositioncomprising a NE and an immunogen comprises two or more adjuvants (See,e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565;WO 99/11241; and WO 94/00153, each of which is hereby incorporated byreference in its entirety).

In some embodiments, a composition comprising a NE and an immunogen ofthe present invention comprises one or more mucoadhesives (See, e.g.,U.S. Pat. App. No. 20050281843, hereby incorporated by reference in itsentirety). The present invention is not limited by the type ofmucoadhesive utilized. Indeed, a variety of mucoadhesives arecontemplated to be useful in the present invention including, but notlimited to, cross-linked derivatives of poly(acrylic acid) (e.g.,carbopol and polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides (e.g., alginate and chitosan), hydroxypropylmethylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose.Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, use of amucoadhesive (e.g., in a composition comprising a NE and immunogen)enhances induction of an immune response in a subject (e.g.,administered a composition of the present invention) due to an increasein duration and/or amount of exposure to an immunogen that a subjectexperiences when a mucoadhesive is used compared to the duration and/oramount of exposure to an immunogen in the absence of using themucoadhesive.

In some embodiments, a composition of the present invention may comprisesterile aqueous preparations. Acceptable vehicles and solvents include,but are not limited to, water, Ringer's solution, phosphate bufferedsaline and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed mineral or non-mineral oil maybe employed including synthetic mono-ordi-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.Carrier formulations suitable for mucosal, subcutaneous, intramuscular,intraperitoneal, intravenous, or administration via other routes may befound in Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa.

A composition comprising a NE and an immunogen of the present inventioncan be used therapeutically (e.g., to enhance an immune response) or asa prophylactic (e.g., for immunization (e.g., to prevent signs orsymptoms of disease)). A composition comprising a NE and an immunogen ofthe present invention can be administered to a subject via a number ofdifferent delivery routes and methods.

For example, the compositions of the present invention can beadministered to a subject (e.g., mucosally (e.g., nasal mucosa, vaginalmucosa, etc.)) by multiple methods, including, but not limited to: beingsuspended in a solution and applied to a surface; being suspended in asolution and sprayed onto a surface using a spray applicator; beingmixed with a mucoadhesive and applied (e.g., sprayed or wiped) onto asurface (e.g., mucosal surface); being placed on or impregnated onto anasal and/or vaginal applicator and applied; being applied by acontrolled-release mechanism; being applied as a liposome; or beingapplied on a polymer.

In some preferred embodiments, compositions of the present invention areadministered mucosally (e.g., using standard techniques; See, e.g.,Remington: The Science and Practice of Pharmacy, Mack PublishingCompany, Easton, Pa., 19th edition, 1995 (e.g., for mucosal deliverytechniques, including intranasal, pulmonary, vaginal and rectaltechniques), as well as European Publication No. 517,565 and Illum etal., J. Controlled Rel., 1994, 29:133-141 (e.g., for techniques ofintranasal administration), each of which is hereby incorporated byreference in its entirety). Alternatively, the compositions of thepresent invention may be administered dermally or transdermally, usingstandard techniques (See, e.g., Remington: The Science arid Practice ofPharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995). Thepresent invention is not limited by the route of administration.

Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, mucosal vaccinationis the preferred route of administration as it has been shown thatmucosal administration of antigens has a greater efficacy of inducingprotective immune responses at mucosal surfaces (e.g., mucosalimmunity), the route of entry of many pathogens. In addition, mucosalvaccination, such as intranasal vaccination, may induce mucosal immunitynot only in the nasal mucosa, but also in distant mucosal sites such asthe genital mucosa (See, e.g., Mestecky, Journal of Clinical Immunology,7:265-276, 1987). More advantageously, in further preferred embodiments,in addition to inducing mucosal immune responses, mucosal vaccinationalso induces systemic immunity. In some embodiments, non-parenteraladministration (e.g., mucosal administration of vaccines) provides anefficient and convenient way to boost systemic immunity (e.g., inducedby parenteral or mucosal vaccination (e.g., in cases where multipleboosts are used to sustain a vigorous systemic immunity)).

In some embodiments, a composition comprising a NE and an immunogen ofthe present invention may be used to protect or treat a subjectsusceptible to, or suffering from, disease by means of administering acomposition of the present invention via a mucosal route (e.g., anoral/alimentary or nasal route). Alternative mucosal routes includeintravaginal and intra-rectal routes. In preferred embodiments of thepresent invention, a nasal route of administration is used, termed“intranasal administration” or “intranasal vaccination” herein. Methodsof intranasal vaccination are well known in the art, including theadministration of a droplet or spray form of the vaccine into thenasopharynx of a subject to be immunized. In some embodiments, anebulized or aerosolized composition comprising a NE and immunogen isprovided. Enteric formulations such as gastro resistant capsules fororal administration, suppositories for rectal or vaginal administrationalso form part of this invention. Compositions of the present inventionmay also be administered via the oral route. Under these circumstances,a composition comprising a NE and an immunogen may comprise apharmaceutically acceptable excipient and/or include alkaline buffers,or enteric capsules. Formulations for nasal delivery may include thosewith dextran or cyclodextran and saponin as an adjuvant.

Compositions of the present invention may also be administered via avaginal route. In such cases, a composition comprising a NE and animmunogen may comprise pharmaceutically acceptable excipients and/oremulsifiers, polymers (e.g., CARBOPOL), and other known stabilizers ofvaginal creams and suppositories. In some embodiments, compositions ofthe present invention are administered via a rectal route. In suchcases, a composition comprising a NE and an immunogen may compriseexcipients and/or waxes and polymers known in the art for forming rectalsuppositories.

In some embodiments, the same route of administration (e.g., mucosaladministration) is chosen for both a priming and boosting vaccination.In some embodiments, multiple routes of administration are utilized(e.g., at the same time, or, alternatively, sequentially) in order tostimulate an immune response (e.g., using a composition comprising a NEand immunogen of the present invention).

For example, in some embodiments, a composition comprising a NE and animmunogen is administered to a mucosal surface of a subject in either apriming or boosting vaccination regime. Alternatively, in someembodiments, a composition comprising a NE and an immunogen isadministered systemically in either a priming or boosting vaccinationregime. In some embodiments, a composition comprising a NE and animmunogen is administered to a subject in a priming vaccination regimenvia mucosal administration and a boosting regimen via systemicadministration. In some embodiments, a composition comprising a NE andan immunogen is administered to a subject in a priming vaccinationregimen via systemic administration and a boosting regimen via mucosaladministration. Examples of systemic routes of administration include,but are not limited to, a parenteral, intramuscular, intradermal,transdermal, subcutaneous, intraperitoneal or intravenousadministration. A composition comprising a NE and an immunogen may beused for both prophylactic and therapeutic purposes.

In some embodiments, compositions of the present invention areadministered by pulmonary delivery. For example, a composition of thepresent invention can be delivered to the lungs of a subject (e.g., ahuman) via inhalation (e.g., thereby traversing across the lungepithelial lining to the blood stream (See, e.g., Adjei, et al.Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J.Pharmaceutics 1990; 63:135-144; Braquet, et al. J. CardiovascularPharmacology 1989 143-146; Hubbard, et al. (1989) Annals of InternalMedicine, Vol. III, pp. 206-212; Smith, et al. J. Clin. Invest. 1989;84:1145-1146; Oswein, et al. “Aerosolization of Proteins”, 1990;Proceedings of Symposium on Respiratory Drug Delivery II Keystone,Colorado; Debs, et al. J. Immunol. 1988; 140:3482-3488; and U.S. Pat.No. 5,284,656 to Platz, et al, each of which are hereby incorporated byreference in its entirety). A method and composition for pulmonarydelivery of drugs for systemic effect is described in U.S. Pat. No.5,451,569 to Wong, et al., hereby incorporated by reference; See alsoU.S. Pat. No. 6,651,655 to Licalsi et al., hereby incorporated byreference in its entirety)).

Further contemplated for use in the practice of this invention are awide range of mechanical devices designed for pulmonary and/or nasalmucosal delivery of pharmaceutical agents including, but not limited to,nebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices suitable for the practice of thisinvention are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis,Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood,Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research TrianglePark, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford,Mass.). All such devices require the use of formulations suitable fordispensing of the therapeutic agent. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants, surfactants, carriers and/or other agents useful in therapy.Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Thus, in some embodiments, a composition comprising a NE and animmunogen of the present invention may be used to protect and/or treat asubject susceptible to, or suffering from, a disease by means ofadministering a compositions comprising a NE and an immunogen bymucosal, intramuscular, intraperitoneal, intradermal, transdermal,pulmonary, intravenous, subcutaneous or other route of administrationdescribed herein. Methods of systemic administration of the vaccinepreparations may include conventional syringes and needles, or devicesdesigned for ballistic delivery of solid vaccines (See, e.g., WO99/27961, hereby incorporated by reference), or needleless pressureliquid jet device (See, e.g., U.S. Pat. No. 4,596,556; U.S. Pat. No.5,993,412, each of which are hereby incorporated by reference), ortransdermal patches (See, e.g., WO 97/48440; WO 98/28037, each of whichare hereby incorporated by reference). The present invention may also beused to enhance the immunogenicity of antigens applied to the skin(transdermal or transcutaneous delivery, See, e.g., WO 98/20734; WO98/28037, each of which are hereby incorporated by reference). Thus, insome embodiments, the present invention provides a delivery device forsystemic administration, pre-filled with the vaccine composition of thepresent invention.

The present invention is not limited by the type of subject administered(e.g., in order to stimulate an immune response (e.g., in order togenerate protective immunity (e.g., mucosal and/or systemic immunity)))a composition of the present invention. Indeed, a wide variety ofsubjects are contemplated to be benefited from administration of acomposition of the present invention. In preferred embodiments, thesubject is a human. In some embodiments, human subjects are of any age(e.g., adults, children, infants, etc.) that have been or are likely tobecome exposed to a microorganism (e.g., Streptococcal bacteria (e.g.,Streptococcus pneumoniae)). In some embodiments, the human subjects aresubjects that are more likely to receive a direct exposure to pathogenicmicroorganisms or that are more likely to display signs and symptoms ofdisease after exposure to a pathogen (e.g., immune suppressed subjects).In some embodiments, the general public is administered (e.g.,vaccinated with) a composition of the present invention (e.g., toprevent the occurrence or spread of disease). For example, in someembodiments, compositions and methods of the present invention areutilized to vaccinate a group of people (e.g., a population of a region,city, state and/or country) for their own health (e.g., to prevent ortreat disease). In some embodiments, the subjects are non-human mammals(e.g., pigs, cattle, goats, horses, sheep, or other livestock; or mice,rats, rabbits or other animal). In some embodiments, compositions andmethods of the present invention are utilized in research settings(e.g., with research animals). In some embodiments, the presentinvention provides a method to elicit an immune response (e.g.,protective immune response) in infants (e.g., from about 0-2 years old)by administering to the infant a safe and effective amount of animmunogenic composition of the invention (e.g., a pediatric vaccine).Further embodiments of the invention include the provision of theimmunogenic S. pneumoniae compositions of the invention for use inmedicine and the use of the S. pneumoniae compositions of the inventionin the manufacture of a medicament for the prevention (or treatment) ofpneumococcal disease.

In yet another embodiment, the present invention is provides a method toelicit an immune response (e.g., a protective immune response) in theelderly population (e.g., in a subject 50 years or over in age,typically over 55 years and more generally over 60 years) byadministering a safe and effective amount of an immunogenic compositionof the invention.

A composition of the present invention may be formulated foradministration by any route, such as mucosal, oral, topical, parenteralor other route described herein. The compositions may be in any one ormore different forms including, but not limited to, tablets, capsules,powders, granules, lozenges, foams, creams or liquid preparations.

Topical formulations of the present invention may be presented as, forinstance, ointments, creams or lotions, foams, and aerosols, and maycontain appropriate conventional additives such as preservatives,solvents (e.g., to assist penetration), and emollients in ointments andcreams.

Topical formulations may also include agents that enhance penetration ofthe active ingredients through the skin. Exemplary agents include abinary combination of N-(hydroxyethyl)pyrrolidone and a cell-envelopedisordering compound, a sugar ester in combination with a sulfoxide orphosphine oxide, and sucrose monooleate, decyl methyl sulfoxide, andalcohol.

Other exemplary materials that increase skin penetration includesurfactants or wetting agents including, but not limited to,polyoxyethylene sorbitan mono-oleoate (Polysorbate 80); sorbitanmono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol polymer (TritonWR-1330); polyoxyethylene sorbitan tri-oleate (Tween 85); dioctyl sodiumsulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); and otherpharmaceutically acceptable surfactants.

In certain embodiments of the invention, compositions may furthercomprise one or more alcohols, zinc-containing compounds, emollients,humectants, thickening and/or gelling agents, neutralizing agents, andsurfactants. Water used in the formulations is preferably deionizedwater having a neutral pH. Additional additives in the topicalformulations include, but are not limited to, silicone fluids, dyes,fragrances, pH adjusters, and vitamins.

Topical formulations may also contain compatible conventional carriers,such as cream or ointment bases and ethanol or oleyl alcohol forlotions. Such carriers may be present as from about 1% up to about 98%of the formulation. The ointment base can comprise one or more ofpetrolatum, mineral oil, ceresin, lanolin alcohol, panthenol, glycerin,bisabolol, cocoa butter and the like.

In some embodiments, pharmaceutical compositions of the presentinvention may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipuritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, preferablydo not unduly interfere with the biological activities of the componentsof the compositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like) that do not deleteriouslyinteract with the NE and immunogen of the formulation. In someembodiments, immunostimulatory compositions of the present invention areadministered in the form of a pharmaceutically acceptable salt. Whenused the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group.

Suitable buffering agents include, but are not limited to, acetic acidand a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid anda salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).Suitable preservatives may include benzalkonium chloride (0.003-0.03%w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) andthimerosal (0.004-0.02% w/v).

In some embodiments, a composition comprising a NE and an immunogen isco-administered with one or more antibiotics. For example, one or moreantibiotics may be administered with, before and/or after administrationof a composition comprising a NE and an immunogen. The present inventionis not limited by the type of antibiotic co-administered. Indeed, avariety of antibiotics may be co-administered including, but not limitedto, β-lactam antibiotics, penicillins (such as natural penicillins,aminopenicillins, penicillinase-resistant penicillins, carboxypenicillins, ureido penicillins), cephalosporins (first generation,second generation, and third generation cephalosporins), and otherβ-lactams (such as imipenem, monobactams), β-lactamase inhibitors,vancomycin, aminoglycosides and spectinomycin, tetracyclines,chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin,metronidazole, polymyxins, doxycycline, quinolones (e.g.,ciprofloxacin), sulfonamides, trimethoprim, and quinolines.

There are an enormous amount of antimicrobial agents currently availablefor use in treating bacterial, fungal and viral infections. For acomprehensive treatise on the general classes of such drugs and theirmechanisms of action, the skilled artisan is referred to Goodman &Gilman's “The Pharmacological Basis of Therapeutics” Eds. Hardman etal., 9th Edition, Pub. McGraw Hill, chapters 43 through 50, 1996,(herein incorporated by reference in its entirety). Generally, theseagents include agents that inhibit cell wall synthesis (e.g.,penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); andthe imidazole antifungal agents (e.g., miconazole, ketoconazole andclotrimazole); agents that act directly to disrupt the cell membrane ofthe microorganism (e.g., detergents such as polmyxin and colistimethateand the antifungals nystatin and amphotericin B); agents that affect theribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol,the tetracyclines, erythromycin and clindamycin); agents that alterprotein synthesis and lead to cell death (e.g., aminoglycosides); agentsthat affect nucleic acid metabolism (e.g., the rifamycins and thequinolones); the antimetabolites (e.g., trimethoprim and sulfonamides);and the nucleic acid analogues such as zidovudine, gangcyclovir,vidarabine, and acyclovir which act to inhibit viral enzymes essentialfor DNA synthesis. Various combinations of antimicrobials may beemployed.

The present invention also includes methods involving co-administrationof a composition comprising a NE and an immunogen with one or moreadditional active and/or immunostimulatory agents (e.g., a compositioncomprising a NE and a different immunogen, an antibiotic, anti-oxidant,etc.). Indeed, it is a further aspect of this invention to providemethods for enhancing prior art immunostimulatory methods (e.g.,immunization methods) and/or pharmaceutical compositions byco-administering a composition of the present invention. Inco-administration procedures, the agents may be administeredconcurrently or sequentially. In one embodiment, the compositionsdescribed herein are administered prior to the other active agent(s).The pharmaceutical formulations and modes of administration may be anyof those described herein. In addition, the two or more co-administeredagents may each be administered using different modes (e.g., routes) ordifferent formulations. The additional agents to be co-administered(e.g., antibiotics, adjuvants, etc.) can be any of the well-known agentsin the art, including, but not limited to, those that are currently inclinical use.

In some embodiments, a composition comprising a NE and immunogen isadministered to a subject via more than one route. For example, asubject that would benefit from having a protective immune response(e.g., immunity) towards a pathogenic microorganism may benefit fromreceiving mucosal administration (e.g., nasal administration or othermucosal routes described herein) and, additionally, receiving one ormore other routes of administration (e.g., parenteral or pulmonaryadministration (e.g., via a nebulizer, inhaler, or other methodsdescribed herein). In some preferred embodiments, administration viamucosal route is sufficient to induce both mucosal as well as systemicimmunity towards an immunogen or organism from which the immunogen isderived. In other embodiments, administration via multiple routes servesto provide both mucosal and systemic immunity. Thus, although anunderstanding of the mechanism is not necessary to practice the presentinvention and the present invention is not limited to any particularmechanism of action, in some embodiments, it is contemplated that asubject administered a composition of the present invention via multipleroutes of administration (e.g., immunization (e.g., mucosal as well asairway or parenteral administration of a composition comprising a NE andimmunogen of the present invention) may have a stronger immune responseto an immunogen than a subject administered a composition via just oneroute.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions, increasing convenience to thesubject and a physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They includepolymer based systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109,hereby incorporated by reference. Delivery systems also includenon-polymer systems that are: lipids including sterols such ascholesterol, cholesterol esters and fatty acids or neutral fats such asmono-di- and tri-glycerides; hydrogel release systems; sylastic systems;peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189,and 5,736,152, each of which is hereby incorporated by reference and (b)diffusional systems in which an active component permeates at acontrolled rate from a polymer such as described in U.S. Pat. Nos.3,854,480, 5,133,974 and 5,407,686, each of which is hereby incorporatedby reference. In addition, pump-based hardware delivery systems can beused, some of which are adapted for implantation.

In preferred embodiments, a composition comprising a NE and an immunogenof the present invention comprises a suitable amount of the immunogen toinduce an immune response in a subject when administered to the subject.In preferred embodiments, the immune response is sufficient to providethe subject protection (e.g., immune protection) against a subsequentexposure to the immunogen or the microorganism (e.g., Streptococcalbacteria (e.g., Streptococcus pneumoniae)) from which the immunogen wasderived. The present invention is not limited by the amount of immunogenused. In some preferred embodiments, the amount of immunogen (e.g.,Streptococcal bacteria (e.g., Streptococcus pneumoniae) or one or morecomponent parts thereof) in a composition comprising a NE and immunogen(e.g., for use as an immunization dose) is selected as that amount whichinduces an immunoprotective response without significant, adverse sideeffects. The amount will vary depending upon which specific immunogen orcombination thereof is/are employed, and can vary from subject tosubject, depending on a number of factors including, but not limited to,the species, age and general condition (e.g., health) of the subject,and the mode of administration.

In some embodiments, each dose (e.g., of a composition comprising a NEand an immunogen (e.g., administered to a subject to induce an immuneresponse (e.g., a protective immune response (e.g., protectiveimmunity))) comprises between about 10⁵ and 10⁸ colony forming units(CFU) of Streptococcus (e.g., Streptococcus pneumoniae) ofkilled/inactivated bacteria, although greater (e.g., about 10⁹, 10¹⁰,10¹¹, 10¹² or more) and lesser (e.g., about 10⁴, 10³, 10² or fewer) CFUof Streptococcus (e.g., Streptococcus pneumoniae) (e.g., killed wholeStreptococcus pneumoniae) may also be utilized. In some embodiments, ananoemulsion solution is utilized to inactivate the Streptococcus (e.g.,Streptococcus pneumoniae). In some embodiments, a nanoemulsion solutionis utilized to inactivate the Streptococcus (e.g., Streptococcuspneumoniae). In some embodiments, the nanoemulsion comprises W₈₀5EC. Insome embodiments, the immunity protects the subject from displayingsigns or symptoms of disease caused by Streptococcus (e.g.,Streptococcus pneumoniae). In some embodiments, the immunity protectsthe subject from challenge with a subsequent exposure to liveStreptococcus (e.g., Streptococcus pneumoniae). In some embodiments,each dose (e.g., administered to a subject to induce and immuneresponse)) comprises between 10 and 10¹⁰ CFU of Streptococcus (e.g.,Streptococcus pneumoniae) per dose. In some embodiments, each dosecomprises between 10⁵ and 10⁸ CFU of Streptococcus (e.g., Streptococcuspneumoniae) per dose. In some embodiments, each dose comprises between10³ and 10⁵ CFU of Streptococcus (e.g., Streptococcus pneumoniae) perdose. In some embodiments, each dose comprises between 10⁵ and 10⁸ CFUof Streptococcus (e.g., Streptococcus pneumoniae) per dose; in someembodiments, each dose comprises 10⁵ CFU of Streptococcus (e.g.,Streptococcus pneumoniae) per dose. In some embodiments, each dosecomprises 10⁶ CFU of Streptococcus (e.g., Streptococcus pneumoniae) perdose. In some embodiments, each dose comprises 10⁷ CFU of Streptococcus(e.g., Streptococcus pneumoniae) per dose. In some embodiments, eachdose comprises more than 10⁸ CFU of Streptococcus (e.g., Streptococcuspneumoniae) per dose. In some preferred embodiments, each dose comprises10⁸ CFU of Streptococcus (e.g., Streptococcus pneumoniae) per dose.

In some embodiments, each dose (e.g., of a composition comprising a NEand an immunogen (e.g., administered to a subject to induce an immuneresponse (e.g., a protective immune response (e.g., protectiveimmunity))) comprises 0.05-5000 μg of an additional immunogen (e.g.,recombinant and/or purified protein, adjuvant (e.g., cholera toxin),etc.). In some embodiments, each dose will comprise 1-500 μg, in someembodiments, each dose will comprise 350-750 μg, in some embodiments,each dose will comprise 50-200m, in some embodiments, each dose willcomprise 25-75 μg of immunogen (e.g., recombinant and/or purifiedprotein). In some embodiments, each dose comprises an amount of theimmunogen sufficient to generate an immune response. An effective amountof the immunogen in a dose need not be quantified, as long as the amountof immunogen generates an immune response in a subject when administeredto the subject.

In some embodiments, it is expected that each dose (e.g., of acomposition comprising a NE and an immunogen (e.g., administered to asubject to induce and immune response)) is from 0.001 to 15% or more(e.g., 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15% or more) by weightimmunogen (e.g., neutralized bacteria, or recombinant and/or purifiedprotein). In some embodiments, an initial or prime administration dosecontains more immunogen than a subsequent boost dose

In some embodiments, when a NE of the present invention is utilized toinactivate a live microorganism, each dose (e.g., administered to asubject to induce and immune response)) comprises between 10 and 10⁹ CFUof the microorganism per dose; in some embodiments, each dose comprisesbetween 10⁵ and 10⁸ CFU of the microorganism per dose; in someembodiments, each dose comprises between 10³ and 10⁵ CFU of themicroorganism per dose; in some embodiments, each dose comprises between10² and 10⁴ CFU of the microorganism per dose; in some embodiments, eachdose comprises 10 CFU of the microorganism per dose; in someembodiments, each dose comprises 10² CFU of the microorganism per dose;and in some embodiments, each dose comprises 10⁴ CFU of themicroorganism per dose. In some embodiments, each dose comprises morethan 10⁹ CFU of the microorganism per dose. In some preferredembodiments, each dose comprises 10³ CFU of the microorganism per dose.

The present invention is not limited by the amount of NE used toinactivate live microorganisms (e.g., Streptococcal bacteria (e.g., S.pneumoniae)). In some embodiments, a 0.1%-5% NE solution is used, insome embodiments, a 5%-20% NE solution is used, in some embodiments, a20% NE solution is used, and in some embodiments, a NE solution greaterthan 20% is used order to inactivate a pathogenic microorganism. Inpreferred embodiments, a 15% NE solution is used.

Similarly, the present invention is not limited by the duration of timea live microorganism is incubated in a NE of the present invention inorder to become inactivated. In some embodiments, the microorganism isincubated for 1-3 hours in NE. In some embodiments, the microorganism isincubated for 3-6 hours in NE. In some embodiments, the microorganism isincubated for more than 6 hours in NE. In preferred embodiments, themicroorganism is incubated for 3 hours in NE (e.g., a 10% NE solution).In some embodiments, the incubation is carried out at 37° C. In someembodiments, the incubation is carried out at a temperature greater thanor less than 37° C. The present invention is also not limited by theamount of microorganism used for inactivation. The amount ofmicroorganism may depend upon a number of factors including, but notlimited to, the total amount of immunogenic composition (e.g., NE andimmunogen) desired, the concentration of solution desired (e.g., priorto dilution for administration), the microorganism and the NE. In somepreferred embodiments, the amount of microorganism used in aninactivation procedure is that amount that produces the desired amountof immunogen (e.g., as described herein) to be administered in a singledose (e.g., diluted from a concentrated stock) to a subject.

In some embodiments, a composition comprising a NE and an immunogen ofthe present invention is formulated in a concentrated dose that can bediluted prior to administration to a subject. For example, dilutions ofa concentrated composition may be administered to a subject such thatthe subject receives any one or more of the specific dosages providedherein. In some embodiments, dilution of a concentrated composition maybe made such that a subject is administered (e.g., in a single dose) acomposition comprising 0.5-50% of the NE and immunogen present in theconcentrated composition. In some preferred embodiments, a subject isadministered in a single dose a composition comprising 1% of the NE andimmunogen present in the concentrated composition. Concentratedcompositions are contemplated to be useful in a setting in which largenumbers of subjects may be administered a composition of the presentinvention (e.g., an immunization clinic, hospital, school, etc.). Insome embodiments, a composition comprising a NE and an immunogen of thepresent invention (e.g., a concentrated composition) is stable at roomtemperature for more than 1 week, in some embodiments for more than 2weeks, in some embodiments for more than 3 weeks, in some embodimentsfor more than 4 weeks, in some embodiments for more than 5 weeks, and insome embodiments for more than 6 weeks.

Generally, the emulsion compositions of the invention will comprise atleast 0.001% to 100%, preferably 0.01 to 90%, of emulsion per ml ofliquid composition. It is envisioned that the formulations may compriseabout 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%,about 0.025%, about 0.05%, about 0.075%, about 0.1%, about 0.25%, about0.5%, about 1.0%, about 2.5%, about 5%, about 7.5%, about 10%, about12.5%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95% or about 98% of emulsion per ml ofliquid composition. It should be understood that a range between any twofigures listed above is specifically contemplated to be encompassedwithin the metes and bounds of the present invention. Some variation indosage will necessarily occur depending on the condition of the specificpathogen and the subject being immunized.

In some embodiments, following an initial administration of acomposition of the present invention (e.g., an initial vaccination), asubject may receive one or more boost administrations (e.g., around 2weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks,around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around4 months, around 6 months, around 9 months, around 1 year, around 2years, around 3 years, around 5 years, around 10 years) subsequent to afirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, and/or more than tenth administration. Although an understandingof the mechanism is not necessary to practice the present invention andthe present invention is not limited to any particular mechanism ofaction, in some embodiments, reintroduction of an immunogen in a boostdose enables vigorous systemic immunity in a subject. The boost can bewith the same formulation given for the primary immune response, or canbe with a different formulation that contains the immunogen. The dosageregimen will also, at least in part, be determined by the need of thesubject and be dependent on the judgment of a practitioner.

Dosage units may be proportionately increased or decreased based onseveral factors including, but not limited to, the weight, age, andhealth status of the subject. In addition, dosage units may be increasedor decreased for subsequent administrations (e.g., boostadministrations).

It is contemplated that the compositions and methods of the presentinvention will find use in various settings, including researchsettings. For example, compositions and methods of the present inventionalso find use in studies of the immune system (e.g., characterization ofadaptive immune responses (e.g., protective immune responses (e.g.,mucosal or systemic immunity))). Uses of the compositions and methodsprovided by the present invention encompass human and non-human subjectsand samples from those subjects, and also encompass researchapplications using these subjects. Compositions and methods of thepresent invention are also useful in studying and optimizingnanoemulsions, immunogens, and other components and for screening fornew components. Thus, it is not intended that the present invention belimited to any particular subject and/or application setting.

The formulations can be tested in vivo in a number of animal modelsdeveloped for the study of mucosal and other routes of delivery. As isreadily apparent, the compositions of the present invention are usefulfor preventing and/or treating a wide variety of diseases and infectionscaused by viruses, bacteria, parasites, and fungi, as well as foreliciting an immune response against a variety of antigens. Not only canthe compositions be used prophylactically or therapeutically, asdescribed above, the compositions can also be used in order to prepareantibodies, both polyclonal and monoclonal (e.g., for diagnosticpurposes), as well as for immunopurification of an antigen of interest.If polyclonal antibodies are desired, a selected mammal, (e.g., mouse,rabbit, goat, horse, etc.) can be immunized with the compositions of thepresent invention. The animal is usually boosted 2-6 weeks later withone or more—administrations of the antigen. Polyclonal antisera can thenbe obtained from the immunized animal and used according to knownprocedures (See, e.g., Jurgens et al., J. Chrom. 1985, 348:363-370).

In some embodiments, the present invention provides a kit comprising acomposition comprising a NE and an immunogen. In some embodiments, thekit further provides a device for administering the composition. Thepresent invention is not limited by the type of device included in thekit. In some embodiments, the device is configured for nasal applicationof the composition of the present invention (e.g., a nasal applicator(e.g., a syringe) or nasal inhaler or nasal mister). In someembodiments, a kit comprises a composition comprising a NE and animmunogen in a concentrated form (e.g., that can be diluted prior toadministration to a subject).

In some embodiments, all kit components are present within a singlecontainer (e.g., vial or tube). In some embodiments, each kit componentis located in a single container (e.g., vial or tube). In someembodiments, one or more kit component are located in a single container(e.g., vial or tube) with other components of the same kit being locatedin a separate container (e.g., vial or tube). In some embodiments, a kitcomprises a buffer. In some embodiments, the kit further comprisesinstructions for use.

EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof. In the experimental disclosure whichfollows, the following abbreviations apply: eq (equivalents); μ(micron); M (Molar); μM (micromolar); mM (millimolar); N (Normal); mol(moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g(grams); mg (milligrams); μg (micrograms); ng (nanograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nM (nanomolar);° C. (degrees Centigrade); and PBS(phosphate buffered saline).

Example 1 Immunogenic Streptococcus pneumoniae Compositions ExperimentalDesign and Materials and Methods.

Outbred CD-1 or C57/B6 (8 groups; 6 mice per group) were intranasallyimmunized with 7.5 μL or 0.1 μL WCPAg in 1, 5, 10 and 20% NE. WCPAg wasgenerated as follows: strain RX1, a capsule-negative mutant derived froma pneumococcus capsular serotype 2 (e.g., an autolysin (lytA)-negativemutant of RX1 (RX1AL⁻)) grown at 37° C. in Todd-Hewitt brothsupplemented with 0.5% yeast extract (THY) and 0.3 μg of erythromycin/mlto about 10⁹ cells/ml, washed and suspended in saline at 10% of theoriginal volume, and then mixed 3:7 (volume/volume) with ethanol, washedand resuspended in saline, and then frozen for later use.

The mice were given a volume of 12 μL (6 μL per nare) delivered manuallyinto the nasal cavity of the mouse. Control groups were: 7.5 μL, WCPAgin PBS, cholera toxin (CT), or Alum (delivered intramuscularly); 0.1 μLWCPAg in PBS; 20% NE alone.

The study design is presented in table 1.

TABLE 1 Design for S. pneumoniae Efficacy Treatment Conc. WCPAg Cells/Volume N Day 0, Week 4, Week 8 (cells/ml) inoculum (μl) (CD1) 7.5 μlWCPAg + 1% NE 1.3 × 10{circumflex over ( )}10 10{circumflex over ( )}812 6 7.5 μl WCPAg + 5% NE 1.3 × 10{circumflex over ( )}10 10{circumflexover ( )}8 12 6 7.5 μl WCPAg + 10% NE 1.3 × 10{circumflex over ( )}1010{circumflex over ( )}8 12 6 7.5 μl WCPAg + 20% NE 1.3 × 10{circumflexover ( )}10 10{circumflex over ( )}8 12 6 0.1 μl WCPAg + 1% NE   l ×10{circumflex over ( )}10 10{circumflex over ( )}6 12 6 0.1 μl WCPAg +5% NE   l × 10{circumflex over ( )}10 10{circumflex over ( )}6 12 6 0.1μl WCPAg + 10% NE   l × 10{circumflex over ( )}10 10{circumflex over( )}6 12 6 0.1 μl WCPAg + 20% NE   l × 10{circumflex over ( )}1010{circumflex over ( )}6 12 6 7.5 μl WCPAg + 1xPBS 1.3 × 10{circumflexover ( )}7  10{circumflex over ( )}8 12 8 20% NE 12 4 7.5 μl WCPAg +Alum IM 1.3 × 10{circumflex over ( )}7  10{circumflex over ( )}8 12 27.5 ml WCPAg + CT 1.3 × 10{circumflex over ( )}7  10{circumflex over( )}8 12 4 0.1 μl WCPAg + 1xPBS   l × 10{circumflex over ( )}1010{circumflex over ( )}6 12 8

Mice were vaccinated at week 0, 4 and 8 (See, e.g., Malley, et al.(2005) Proc Natl Acad Sci USA. March 29; 102(13):4848-53). Blood sampleswere obtained via saphenous vein bleed at weeks 0, 2, 4, 6, 8, and 10.IgG antibodies against the lysed whole bacterium were assayed via ELISAafter each bleed week.

At week 12, mice were given rifampin subcutaneously to clear any nasalcolonization. Seven days post rifampin, mice were challenged with 1×10⁵CFU live S. pneumoniae/mouse and monitored daily for weight andtemperature. On day 7 after challenge, the mice were sacrificed andcardiac bleed was taken for CBC and IgG titers, heads were preserved forhistology and retrograde nasophyringeal wash was taken for S. pneumoniaecolony counts on blood-agar plates. IgG end-titer ELISA was performedin-house using standard assays known in the art. CBC was performed atthe University of Michigan Unit for Laboratory Animal Medicine (ULAM)per standard procedures known in the art

Test Formulation. Vaccine formulations were prepared by vigorouslymixing WCPAg and NE or Alum or CT in 10×PBS and sterile water forinjection for about 20 seconds just prior to immunization (30 to 60minutes) (See FIG. 1 for formulations).

Immunization Procedures. Mice were vaccinated intranasally (I.N.) orintramuscularly (I.M.) with 3 administrations of vaccine and antibodyresponses were measured at two week intervals over a period of 12 weeks.For I.N. immunization, animals were anaesthetized with Isoflurane(IMPAC6) and held in an inverted position until 6 μL of vaccine,delivered with a pipette tip, were completely inhaled. For I.M.vaccination, mice were anaesthetized with Isoflurane (IMPAC6) and 124 ofWCPAg/Alum vaccine was injected into the epaxial muscle.

Blood Sample Collection. Blood samples were obtained from the saphenousvein at various time points during the course of the trials. The finalsample was obtained by cardiac puncture from euthanized, premorbid mice.Serum was separated from the blood by centrifugation at 1500×g for 5minutes after coagulation. Serum was stored at −20° C. until used forELISA. For CBC, blood was collected after cardiac puncture and placedinto heparinized tubes with continuous rocking until delivery to ULAM.

Weight/Temperature Monitoring. Weight and body core temperature (rectalthermometer BAT-12) temperatures were taken 5-7 days after challenge.

Rifampin Administration. Two weeks following the last immunization,animals in all groups received 1 mg of rifampin subcutaneously on twoconsecutive days to eliminate pneumococcal colonization.

Retrograde Nasopharyngeal Wash. Nasopharyngeal wash was obtained frommice euthanized by Isoflurane inhalation. After the trachea wasdissected, a 22-gauge catheter (Angiocath, B-D) attached to a 1 mlsyringe was inserted into the trachea further to the nasal cavity. Thenares of the mice were washed with 1 μL PBS and solution was let to fallonto a blood agar plate coated with gentamycin.

Analysis of IgG titers: ELISA for anti-S. pneumoniae antibodies wasutilized to determine IgG titers. Preparation of WCPAg-coated ELISAPlates. Coating buffer, 0.05M carbonate-bicarbonate buffer (pH 9.6), wasmade from carbonate-bicarbonate buffer capsules (SIGMA) by dissolvingthe in double distilled water according to manufacturer's instructions.S. pneumoniae lysate (whole bacterium lysed in non-ionic lysis buffer)was diluted in coating buffer and 100 μL per well was added to 96-wellplates (Nunc, Maxisorb Plates). Plates were incubated overnight and keptat 4° C. until used. Stored plates were warmed up ½ hour at 37° C. andantigen solution was removed by inversion and tamping on paper towels.The plates were blocked with 1% milk in PBS (1004 per well) andincubated at 1 hour at 37° C. Blocking solution was removed from wellsjust before diluted serum was added.

Preparation of the primary antibody dilutions. Mouse sera was diluted in0.1% BSA in PBS. 1004 of the diluted serum was added per well induplicate and incubated overnight at 4° C. The next day, the plates werewarmed for ½ hour at 37° C. The serum dilutions were removed byinversion/tamping and washed 3× with ELISA wash buffer (Quantikine, R&DSystems).

Preparation of a recommended dilution of secondary antibody conjugate.Secondary antibody (goat anti-mouse F(c) alkaline-phosphatase conjugated(Rockland) was diluted at 1:1000 in 0.1% BSA in PBS. 100 μL of thediluted secondary antibody was added to each well and incubated 1 hourat 37° C. The secondary antibody was removed by inversion/tamping andthe plate washed 3 times with ELISA wash buffer (Quantikine, R&DSystems).

Preparation of alkaline phosphatase substrate solution. SigmafastP-Nitrophenyl Phosphate Tablets (pNPP, SIGMA) were dissolved in doubledistilled water according to the manufacturer's recommendations. Afterremoving the last wash, 100 μL of pNPP solution was added to each wellincubated and read every ½ hour until saturation was achieved. Thetimepoint closest to saturation was chosen as analysis time.

End-point determination. The antibody endpoint titers are defined as thereciprocal of the highest serum dilution which gives a reading abovecutoff value determined by the dilution of control sera and platebackground (passes at least two standard deviations above average forbackground wells, See, e.g., Frey, et al. (1998) Journal ofImmunological Methods 221:35-41; Classen, et al. (1987) Journal ofClinical Microbiology. 25:600).

Example 2 Distribution of Anti-S. pneumoniae Antibody Titers at 8 Weeksafter Two Vaccinations

FIG. 2 shows anti-S. pneumoniae IgG titer distributions at week 8, aftertwo intranasal vaccine doses given one month apart on weeks 0 and 4, forexperimental and control groups. All mice vaccinated with 10⁸ CFU WCPAgplus varying concentrations of nanoemulsion obtained a serum antibodytiter of 5×10³ or greater, with a maximum of 10⁵ and an average acrossall groups of 7×10⁵. Titers for individual animals (circles) and meanserum titer per group (dash) are shown (See FIG. 2).

Example 3 Distribution of Anti-S. pneumoniae Antibody Titers at 10 Weeksafter Three Vaccinations

FIG. 3 shows Anti-S. pneumoniae IgG titer distributions at week 10,after three intranasal vaccine doses given one month apart on weeks 0,4, and 8 for experimental and control groups. All mice vaccinated with10⁸ CFU WCPAg plus varying concentrations of nanoemulsion obtained aserum antibody titer of 5×10³ or greater, with a maximum of 5×10⁶ and anaverage across all 10⁸ CFU/vaccine groups of 4×10⁵. Titers forindividual animals (circles) and mean serum titer per group (dash) areshown (See FIG. 3).

Example 4 Nanoemulsion Utilized to Adjuvant Ethanol Killed Whole Cell S.pneumoniae

Unless otherwise described herein, materials and methods indicated inExample 1 were utilized. Ethanol inactivated whole cell S. pneumoniaeantigen (either 10⁶ or 10⁸ cells) was mixed with W₈₀5EC nanoemulsion(ranging from 1% to 20%). This mixture was used to intranasallyvaccinate (6 μl/nare) 8 week old outbred CD-1 mice (Jackson Labs, Bararbor, ME). The mice were vaccinated and then boosted at 4 weeksfollowing prime vaccination. The mice were nasally inoculated with 10⁸live wildtype strain 6B S. pneumoniae at 11 weeks and sacrificed forcolony enumeration at 12 weeks. Serum anti-pneumococcal IgG titers, asmeasured by ELISA, were found to be 1 to 1.5 logs greater than negativecontrols and approached within 0.5 log of positive (alum) control (SeeFIG. 4). The high serum titers in the high antigen dose group correlatedwith increased ability to eradicate intranasal colonization followingchallenge with wild-type S. pneumoniae (See FIG. 5).

Example 5 Compositions and Methods Utilizing NanoemulsionInactivated/Killed Whole Cell S. pneumoniae

Nanoemulsion was utilized to kill and/or inactivate live S. pneumoniae,which was then subsequently administered to subjects to generate immuneresponse to nanoemulsion killed S. pneumoniae compositions. In order toevaluate the microbiocidal activity of nanoemulsion against S.pneumoniae, 1×10⁸ live, acapsular LytA-S. pneumoniae mutant was mixedwith varying concentrations of nanoemulsion (W₈₀5EC, 1%, 5%, 10% or20%). The bacteria were incubated with NE for 30 minutes, 1 hour or 3hours. The nanoemulsion was separated by centrifugation and thekilled/inactivated whole cell acapsular LytA-S. pneumoniae pellets werewashed to remove any remaining NE. Resuspended pellets were plated onblood agar for colony enumeration. Complete inactivation of the S.pneumoniae was noted at all time points (See FIG. 6).

A composition comprising nanoemulsion killed S. pneumoniae was utilizedto generate immune response in subjects. Two different concentrations ofnanoemulsion (5% and 15%) were utilized to to inactivate 10⁷ or 10⁹live, acapsular LytA-S. pneumoniae mutant cells. For comparison,bacteria were also inactivated with ethanol (EI). Inactivation wasverified using plate culture.

Following the inactivation procedure, mice were immunized with thecombined inactivated S. pneumoniae and NE. Immunizations were deliveredintranasally except for positive (Alum) control. The mice were primedand then boosted at 4 weeks (See FIG. 7). The mice were nasallyinoculated with 10⁸ live wildtype strain 6B S. pneumoniae at 8 weeks andsacrificed for colony enumeration at 9 weeks. Serum anti-pneumococcalIgG titers were observed that approached 10⁵ in mice vaccinated with 15%NE-10⁹ inactivated bacteria. These titers were equivalent or greaterthan (alum) control mice (See FIG. 8). The high serum titers in the highantigen dose group correlated with increased ability to eradicateintranasal colonization following challenge with wild-type S. pneumoniae(See FIG. 9).

Thus, in some embodiments, the present invention provide that nasaladministration of a whole cell Streptococcus pneumoniae antigen (WCPAg,killed and/or inactivated by mixing with ethanol and/or nanoemulsion)mixed with nanoemulsion induces IgG response and eradicates upperrespiratory colonization.

Example 6 Identification of S. pneumoniae Immunogens

Although an understanding of a mechanism is not necessary to practicethe present invention, and the invention is not limited to anyparticular mechanism of action, experiments were conducted duringdevelopment of embodiments of the invention in order to furthercharacterize and/or identify immunoreactive proteins present ininactivated S. pneumoniae, (e.g., inactivated using nanoemulsion orethanol).

Experiments were designed to identify potential protective antigens.Work involved identification of immunoreactive proteins via westernblotting. NE inactivated S. pneumoniae, ethanol inactived S. pneumoniae,and wildtype 6B inactivated protein were electrophoretically separatedand probed with either serum from mice vaccinated with 15% NE-10⁹ S.pneumoniae or 0.5 mg/kg Alum-S. pneumoniae (See FIG. 10). Bands atseveral molecular weights corresponding to known conserved orsemi-conserved pneumococcal proteins were identified (e.g., PsaA, PiuA,PavA).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described compositions and methods of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe present invention.

1. An immunogenic composition comprising a nanoemulsion and aStreptococcus antigen.
 2. The immunogenic composition of claim 1 whereinthe Streptococcus antigen comprises killed whole Streptococcuspneumoniae cells.
 3. The immunogenic composition of claim 2, wherein theStreptococcus pneumoniae cells are killed via mixing with a nanoemulsionor alcohol.
 4. The immunogenic composition of claim 3, wherein thealcohol is ethanol.
 5. The immunogenic composition of claim 2, whereinsaid composition comprises 10⁶ CFU of killed Streptococcus pneumoniaecells.
 6. The immunogenic composition of claim 2, wherein saidcomposition comprises 10⁸ CFU of killed Streptococcus pneumoniae cells.7. The immunogenic composition of claim 1, wherein the nanoemulsioncomprises a non-ionic surfactant, ethanol, cetylpyridinium chloride(CPC), oil and water.
 8. The immunogenic composition of claim 1, whereinthe composition comprises between 1-20% nanoemulsion solution.
 9. Theimmunogenic composition of claim 1, wherein said composition is heatstable.
 10. The immunogenic composition of claim 1, further comprising apharmaceutically acceptable carrier.
 11. The immunogenic composition ofclaim 1, further comprising an adjuvant.
 12. The immunogenic compositionof claim 11, wherein the adjuvant is cholera toxin
 13. A method ofinducing an immune response to Streptococcus pneumoniae in a subjectcomprising: a) providing an immunogenic composition comprising ananoemulsion and an immunogen, wherein said immunogen comprises killedwhole cell Streptococcus pneumoniae; and b) administering saidcomposition to said subject under conditions such that said subjectgenerates an immune response to Streptococcus pneumoniae .
 14. Themethod of claim 13, wherein said administering comprises contacting amucosal surface of said subject with said composition.
 15. The method ofclaim 14, wherein said mucosal surface comprises nasal mucosa. 16-17.(canceled)
 18. The method of claim 13, wherein said immune responsecomprises a systemic IgG response to Streptococcus pneumoniae in saidsubject.
 19. The method of claim 13, wherein said composition isadministered to said subject under conditions such that between 10⁶ and10⁸ CFU of killed Streptococcus pneumoniae is present in a doseadministered to said subject.
 20. The method of claim 13, wherein saidnanoemulsion comprises a non-ionic surfactant, ethanol, cetylpyridiniumchloride (CPC), oil and water.
 21. The method of claim 13, wherein saidimmune response protects said subject from displaying signs or symptomsof disease caused by Streptococcus pneumoniae .
 22. The method of claim13, wherein said immune response protects said subject from challengewith a subsequent exposure to live Streptococcus pneumoniae . 23-24.(canceled)